Methods of treatment using anti-human IL-21 monoclonal antibodies

ABSTRACT

Human anti-human IL-21 monoclonal antibodies and the hybridomas that produce them are presented. Certain of these antibodies have the ability to bind native human IL-21, a mutant recombinant IL-21 protein and/or peptide regions of human IL-21. These human anti-IL-21  antibodies are useful in therapeutic treatment of autoimmune and inflammatory diseases, particularly diseases mediated by T follicular helper cells, B cells, T H  cells or T H 17 cells.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation of Ser. No. 13/348,366 filed Jan. 11,2012 now U.S. Pat. No. 8,226,948 B1, issued on Jul. 24, 2012, which is acontinuation of Ser. No. 12/483,098 filed Jun. 11, 2009 now U.S. Pat.No. 8,124,089, issued Feb. 28, 2012, which is a continuation in part ofSer. No. 12/330,334 filed Dec. 8, 2008 now U.S. Pat. No. 7,883,700,issued Feb. 8, 2011, which claims the benefit of U.S. ProvisionalApplication Ser. No. 61/012,329, filed Dec. 7, 2007, the entire contentsof which are herein incorporated by reference.

BACKGROUND OF THE INVENTION

The immune system is the body's primary defense against diseases causedby pathogens, namely bacteria, viruses, fungi etc, as well as againstdiseases caused by abnormal growth of the body's own cells and tissues(i.e. cancerous tumors). Normally, the immune system is able todistinguish between the body's normal cells or “self” and foreignpathogens or abnormal cells or “non-self”. The processes by which theimmune system refrains from reacting to one's own body is calledtolerance. Sometimes, the immune system loses the ability to recognize“self” as normal and the subsequent response directed against the tissueor cells, results in loss of tolerance, a state of autoimmunity. Thepathologies resulting from autoimmunity often have serious clinicalconsequences and are one of the major health problems in the world,especially in developed nations.

IL-21 is a potent immunomodulatory four-α-helical bundle type I cytokinethat binds to a heterodimeric receptor composed of IL-21R and the commongamma chain (reviewed by Spolski and Leonard, Annu Rev Immunol. Nov. 8,2007). IL-21 is produced by NK-T and CD4+ T cells (includingpro-inflammatory Th17 cells and follicular helper T_(FH) cells that areimportant for germinal center responses) and has pleiotropic effects onboth innate and adaptive immune responses, including enhancedproliferation of B and T cells, increased cytotoxicity of CD8+ T cellsand natural killer (NK) cells, differentiation of B cells intoimmunoglobulin-secreting plasma cells, and regulation of the Th17 celllineage (see below). IL-21 can also inhibit the antigen-presentationfunction of dendritic cells and can induce apoptosis in B cells and NKcells under certain conditions. IL-21 has potent anti-tumor activity,but has also been associated with the development of various autoimmunediseases, including systemic lupus erythematosus (SLE), rheumatoidarthritis (RA), inflammatory bowel disease (IBD) and psoriasis (reviewedby Spolski and Leonard, Annu Rev Immunol. Nov. 8, 2007).

IL-21 has been shown to modulate antibody responses by directly actingon B cells. (Mehta et al., J. Immunol., 170:4111-4118, 2003; Ozaki etal., Science, 298:1630-1634, 2002; Suto et al., Blood, 100:4565-4573,2002). IL-21 can induce the differentiation of naïve human B cells intoantibody-secreting plasma cells (Ozaki et al. J. Immunol. 173:5361,2004; Ettinger et al., J. Immunol. 175:7867-79, 2005; Ettinger et al, J.Immunol. 178:2872-82, 2007; Kuchen et al. J. Immunol. 179:5886-96, 2007)and to stimulate the production of IgE in human B cell (Kobayashi et al.Human Immunol. doi:10:1016/j.humimm.2008.10.) In IL-21 or IL-21Rdeficient animals, fewer antibody-secreting cells are generated from thegerminal center reaction and affinity maturation is reduced (Zotos etal., submitted). Extrafollicular antibody forming cells, which areimplicated in autoimmunity, require cognate help from a subset ofspecialized CD4 T cells that secrete IL-21 (Odegard, et al., JEM205(12):2873-2886, 2008).

Generation of antibodies against allogenic MHC is a pivotal phenomenonin transplant rejection. Transplant recipients who develop titres ofanti-MHC antibodies (highly sensitized transplant patients) are t riskfor chronic rejection and are poor candidates for new grafts due tolikelihood of antibody mediated rejection of the new transplant (Smith,et al., Am J Transplantation 8: 1-11, 2008). In a rat model of acuterenal allograft rejection, IL-21 and IL-21R were uniquely increased inintravascular mononuclear cells of renal allografts but not isografts(Hecker, et al., Immunobiology: doi:10.1016/j.imbio.2008.04.004,(2008)). In human cardiac transplants undergoing rejection, expressionlevels of IL-21 and IL-21R correlate with the ISHLT rejection grade, andhighest expression is present in grades 1R and 2R (Baan, et al.,Transplantation 83(11): 1485-1492, 2007).

In graft-versus-host-disease (GVHD), the anti-allo response is mediatedby uncontrolled activation of T lymphocytes from the graft, which directan inflammatory response against host tissues. Regulatory T cells (Treg)can modulate this response in animal models. IL-21 has been shown tocounteract the regulatory functions of Treg (Clough et al., J Immunol180: 5395-5401, 2008). In mouse models of GVHD, transfer of IL-21deficient T cells resulted in significantly reduced clinical signs andhistological scores and increased survival, compared with WT T cells.Decreased frequency of IFN-gamma secreting T cells and increased Tregswere observed in the colon mucosa. IL-21 blockade using anti-mIL-21 mAband WT T cell transfer produced similar results (Bucher et al., Blood(ASH Annual Meeting Abstracts) 2008 112: Abstract #2342).

It has also recently been shown that IL-21 is both produced by andrequired for the differentiation of mouse pro-inflammatory Th17 cells(Korn et al. Nature. 448:484-487, 2007; Nurieva et al. Nature448:480-483, 2007; Zhou et al., Nat Immunol. 8:967-974, 2007; Wei et al.J Biol. Chem. 282:34605-34610, 2007). Human Th17 cells also produceIL-21 and studies are ongoing to determine whether IL-21 acts as anautocrine factor for human Th17 cells, as it does for mouse Th17 cells.Ozaki et al. (J. Immunol. 173:5361, 2004) demonstrated that IL-21expression is elevated in lupus-prone BXSB-Yaa mice, a model forsystemic lupus erythematosus (SLE), at an age when the earlycharacteristics of autoimmune processes first become evident. Treatmentof these BXSB-Yaa mice with a soluble mouse IL-21 receptor (mIL-21R-Fc)partially inhibits various disease parameters, includingglomerulonephritis (Bubier et al., Ann N Y Acad Sci. 1110:590-601,2007). Treatment with mIL-21R-Fc has also been shown to be efficaciousin another pre-clinical disease model of SLE, the MRL/lpr mouse (Herberet al. J. Immunol. 178: 3822-3830, 2007), as well as in thecollagen-induced arthritis (CIA) model of rheumatoid arthritis (Young etal., Arthr Rheum 56:1152-1163, 2007). Preliminary human data alsosuggest dysregulation of IL-21 and IL-21R in SLE (Mitoma et al. Int JMol Med. 16:609-615, 2005; Wang et al., Chinese J. Cell. Mol. Immunol.23(11):1041-1042, 2007; Sawalha et al. Ann Rheum Dis 67: 458-461, 2008).More recently, Rus et al. data obtained from 24 SLE patients and 15healthy controls (Nguyen et al., ACR/ARHP Scientific Meeting, 1760/482,2008 Oct. 24-29 San Francisco, Calif.). Rus et al. showed that 1) IL-21mRNA expression is significantly increased in CD4+ T cells from lupuspatients compared to controls, 2) IL-21 levels are significantlyelevated in sera from patients with active compared to inactive SLE orcontrols, 3) IL-21 enhances CD4+ T cells and CD19+ B cells proliferationin patients and controls in a dose dependent fashion, 4) IL-21 enhancesanti-CD40 induced plasma cell differentiation in normal controls and SLEpatients, and 5) elevated levels of IL-21 may contribute toproliferation of autoreactive CD4+ T cells and plasma celldifferentiation in SLE.

Monteleone et al have demonstrated that IL-21 RNA and protein expressionis increased in inflamed but not uninflamed tissue from Crohn's disease(CD) (and, to a lesser degree, ulcerative colitis) patients and thatIL-21 production by CD3+ cells from lamina propria mononuclear cellsfrom CD patients is also enhanced (Monteleone et al. Gastroenterology128:687-694, 2005; Monteleone et al. Gut 55:1774-1780, 2006; Peluso etal., J Immunol 178:732-739, 2007). These authors suggested that IL-21regulates experimental colitis by modulating the balance betweenregulatory T cells (Tregs) and Th17 cells (Fantini et al. Eur. J.Immunol. 37:3155-3163, 2007). Inhibition of IL-21 in vivo with a solubleIL-21 receptor in either mouse or rat models of colitis leads tosignificant reductions in clinical signs of colitis (Young et al. US2006/0039902).

The IL-21 receptor is expressed by NK cells, and NK cells have beenshown to be responsive to treatment with IL-21 both in vivo and invitro. In oncology patients treated with recombinant human IL-21,altered recirculation patterns in lymphocyte subsets including NK cells,and increased expression of markers of NK cell activation and cytolyticeffector capacity were observed (Frederiksen, et al., Cancer ImmunolImmunother 57(10): 1439-1449, 2008). In autoimmune diseases, NK cellactivity may play a role in promoting inflammation and associated tissuedamage. Tissue homing of NK cells is directed by chemoattractantsreleased at the site of inflammation (Morris and Ley, Curr Mol. Med.;4(4):431-8, 2004). Lamina propria NK cells from patients with Crohn'sDisease released greater quantities of IFN-γ and TNF-α when stimulatedin vitro with IL-21 and IgG, compared with LPNK cells from controls (Liuand Jiu, Chronic Inflammation of Liver and Gut, Falk Symposium abst. No.163, 2008 Mar. 14-15). NK cells are also reported to regulateautoimmunity and transplant rejection through their interactions withdendritic cells (DC), by killing immature or activated DC, and byreleasing cytokines that affect the activation state and antigenpresentation functions of the DC (Vivier et al., Nat Immunol9(5):503-510, 2008; Laffont et al., Blood 112:661-671, 2008). Acomparison of peripheral blood mononuclear cells from tolerant andnon-tolerant liver allograft recipients showed changes in thetranscriptional program of NK cells (Martinez-Llordella et al., J ClinInvest 118(8):2845-2857, 2008). Thus, blockade of IL-21 may modulate theactivation status of NK cells, reduce their contribution to tissueinflammation in autoimmune diseases, and alter the clinical course oftransplant rejection. NK cells are also reported to regulateautoimmunity and transplant rejection through their interactions withdendritic cells (DC), by killing immature or activated DC and byreleasing cytokines that affect the activation state and alter antigenpresentation functions of the DC. Thus, blockade of IL-21 may modulatethe activation of NK cells and reduce their contribution to tissueinflammation in autoimmune diseases.

The present invention provides anti-human IL-21 monoclonal antibodiesand methods of using those antibodies that inhibit the symptoms andbiological activities that manifest as autoimmune and inflammatorydisorders and are associated with IL-21/IL-21 receptor interactions.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is an alignment based on the Kabat numbering system of amino acidresidues comprising the variable heavy chain regions of antibodiesdesignated by clone numbers 362.78.1.44 (78), 362.597.3 (597),362.75.1.1 (75), 366.552.11 (552), 366.328.10 (328), and correspondingwith SEQ ID NO: 29, SEQ ID NO: 45, SEQ ID NO: 13, SEQ ID NO: 77 and SEQID NO: 61, respectively.

FIG. 2 is an alignment based on the Kabat numbering system of aminoresidues comprising the variable light chain region of antibodiesdesignated by clone numbers 362.78.1.44 (78), 362.597.3 (597),362.75.1.1 (75), 366.552.11 (552), 366.328.10 (328) and correspondingwith SEQ ID NO: 37, SEQ ID NO: 53, SEQ ID NO: 21, SEQ ID NO: 85 and SEQID NO: 69, respectively.

FIG. 3 illustrates MALDI/TOF mass spectra of IL-21 peptide sequenceregions obtained from IL-21 alone and the IL-21 immune complex. IL-21peptide sequences, EKKPPKEF (SEQ ID NO: 2 from residue 129 to 136) (m/z,1002.5619 Da) and LERFKSLL (SEQ ID NO: 2 from residue 137 to 144) (m/z,1005.6091 Da) of the free-state of IL-21 (A). Peptide mass shifting dueto the retention of amide deuteration in the presence of IL-21 mAb (B).Another IL-21 peptide sequence region, KSLLQKMIHQHLSSRTHGSEDS (SEQ IDNO: 2 from residue 141 to 162) (m/z, 2519.2451) of the free-state ofIL-21 (C). A partial peptide mass shifting due to the retention of amidedeuteration in the presence of IL-21 mAb (D).

FIG. 4 illustrates selected ion chromatograms of acetylated andnon-acetylated peptides. Selected single ion chromatogram of acetylatedTCPSCDSYEKKPPKEF (SEQ ID NO: 2 from residue 119 to 136) (m/z, 1986 Da)isolated from IL-21 alone (A) and the same chromatographic trace of theIL-21 immune complex (B). The embedded mass spectrum is triply chargedstate of the peptide mass (m/z, 662.9). The third trace shows theselected ion chromatogram of the peptide ion at m/z 1018 Da, which isacetylated KSLLQKMI (SEQ ID NO: 2 from residue 141 to 148) isolated fromIL-21 alone (C) and the same chromatographic trace of the IL-21 immunecomplex (ID). The embedded mass spectrum is doubly charged state of thepeptide mass (m/z, 509.1)

FIGS. 5A-5C depict tetravalent, bispecific Fc fusion and Mab formatshaving Fv regions with specificity for two different targets (referredto herein as targets X and Y). Fv domains against target X are indicatedby a striped fill, Fv domains against target Y are indicated by a grayfill, and the Ig constant domains are indicated by stippled fill. FIG.5A shows a tandem single chain Fv Fc fusion (tascFv-Fc); FIG. 5B shows abi-single chain Fv Fc fusion (biscFv-Fc); and FIG. 5C shows a wholemonoclonal antibody with a single chain Fv (scFv) fused to the carboxylterminus (BiAb).

BRIEF DESCRIPTION OF THE INVENTION

In one aspect, the present invention provides an anti-human IL-21monoclonal antibody comprising at least 80% identity to amino acidresidues 20 to 145 of SEQ ID NO: 29 and at least 80% identity to aminoacid residues 21 to 126 of SEQ ID NO: 37. In certain embodiments, theantibodies comprise changes of at least 80% identity that are in theheavy chain variable region CDR1 of SEQ ID NO: 31.

In another aspect, the present invention provides an anti-human IL-21monoclonal antibody comprising: (a) a heavy chain region comprising: (i)a heavy chain variable region CDR1 comprising SEQ ID NO: 31; (ii) aheavy chain variable region CDR2 comprising SEQ ID NO: 33; and (iii) aheavy chain variable region CDR3 comprising SEQ ID NO: 35; and (b) alight chain region comprising: (i) a light chain variable region CDR1comprising SEQ ID NO: 39; (ii) a light chain variable region CDR2comprising SEQ ID NO: 41; and (iii) a light chain variable region CDR3comprising SEQ ID NO: 43. In certain embodiments, the invention providesan anti-human IL-21 monoclonal antibody comprising amino acids residues20 to 145 of SEQ ID NO: 29 and amino acid residues 21 to 126 of SEQ IDNO: 37. In other embodiments, the antibody is further comprising aminoacid residues 1 to 145 of SEQ ID NO: 29 and amino acid residues 1 to 126of SEQ ID NO: 37. Another embodiment of the present invention provides ahybridoma designated 362.78.1.44, wherein the hybridoma is depositedwith the American Type Culture Collection having the ATCC Patent DepositDesignation PTA-8790, and the invention includes the antibody producedby the hybridoma.

Another aspect of the present invention provides an anti-human IL-21monoclonal antibody comprising: (a) a heavy chain region comprising: (i)a heavy chain variable region CDR1 comprising SEQ ID NO: 47; (ii) aheavy chain variable region CDR2 comprising SEQ ID NO: 49; and (iii) aheavy chain variable region CDR3 comprising SEQ ID NO: 51; and (b) alight chain region comprising: (i) a light chain variable region CDR1comprising SEQ ID NO: 55; (ii) a light chain variable region CDR2comprising SEQ ID NO: 57; and (iii) a light chain variable region CDR3comprising SEQ ID NO: 59. In certain embodiments, the invention providesan anti-human IL-21 monoclonal antibody comprising amino acids residues20 to 145 of SEQ ID NO: 45 and amino acid residues 21 to 126 of SEQ IDNO: 53. In other embodiments, the invention is further comprising aminoacid residues 1 to 145 of SEQ ID NO: 45 and amino acid residues 21 to126 of SEQ ID NO: 53. Another embodiment of the present inventionprovides a hybridoma designated 362.597.3, wherein the hybridoma isdeposited with the American Type Culture Collection having the ATCCPatent Deposit Designation PTA-8786, and the invention includes theantibody produced by the hybridoma.

In another aspect, the present invention provides an anti-human IL-21monoclonal antibody comprising at least 80% identity to amino acidresidues 20 to 141 of SEQ ID NO: 13 and at least 80% identity to aminoacid residues 21 to 126 of SEQ ID NO: 21. In one embodiment, theinvention includes a monoclonal antibody where any amino acid changesare conservative amino acid changes.

In another aspect, the present invention provides an anti-human IL-21monoclonal antibody comprising: (a) a heavy chain region comprising: (i)a heavy chain variable region CDR1 comprising SEQ ID NO: 15; (ii) aheavy chain variable region CDR2 comprising SEQ ID NO: 17; and (iii) aheavy chain variable region CDR3 comprising SEQ ID NO: 19; and (b) alight chain region comprising: (i) a light chain variable region CDR1comprising SEQ ID NO: 23; (ii) a light chain variable region CDR2comprising SEQ ID NO: 25; and (iii) a light chain variable region CDR3comprising SEQ ID NO: 27. In certain embodiments, the present inventionincludes an anti-human IL-21 monoclonal antibody comprising amino acidresidues 20 to 141 of SEQ ID NO: 13 and amino acid residues 21 to 126 ofSEQ ID NO: 21. In another embodiment, the invention is furthercomprising amino acid residues 1 to 141 of SEQ ID NO: 13 and amino acidresidues 1 to 126 of SEQ ID NO: 21. Another embodiment of the presentinvention provides a hybridoma designated 362.75.1.1, wherein thehybridoma is deposited with the American Type Culture Collection havingthe ATCC Patent Deposit Designation PTA-8791, and the antibody producedby the hybridoma.

In another aspect, the present invention provides an anti-human IL-21monoclonal antibody comprising: (a) a heavy chain region comprising: (i)a heavy chain variable region CDR1 comprising SEQ ID NO: 79; (ii) aheavy chain variable region CDR2 comprising SEQ ID NO: 81; and (iii) aheavy chain variable region CDR3 comprising SEQ ID NO: 83; and (b) alight chain region comprising: (i) a light chain variable region CDR1comprising SEQ ID NO: 87; (ii) a light chain variable region CDR2comprising SEQ ID NO: 89; and (iii) a light chain variable region CDR3comprising SEQ ID NO: 91. In one embodiment, the present inventionprovides an anti-human IL-21 monoclonal antibody comprising amino acidsresidues 20 to 136 of SEQ ID NO: 77 and amino acid residues 23 to 129 ofSEQ ID NO: 85. In another embodiment, the invention is furthercomprising amino acid residues 1 to 136 of SEQ ID NO: 77 and amino acidresidues 1 to 129 of SEQ ID NO: 85. Another embodiment of the presentinvention provides a hybridoma designated 366.552.11, wherein thehybridoma is deposited with the American Type Culture Collection havingthe ATCC Patent Deposit Designation PTA-8787, and the antibody producedby the hybridoma.

In another aspect, the present invention provides an anti-human IL-21monoclonal antibody comprising: (a) a heavy chain region comprising: (i)a heavy chain variable region CDR1 comprising SEQ ID NO: 63; (ii) aheavy chain variable region CDR2 comprising SEQ ID NO: 65; and (iii) aheavy chain variable region CDR3 comprising SEQ ID NO: 67; and (b) alight chain region comprising: (i) a light chain variable region CDR1comprising SEQ ID NO: 71; (ii) a light chain variable region CDR2comprising SEQ ID NO: 73; and (iii) a light chain variable region CDR3comprising SEQ ID NO: 75. In certain embodiments, the present inventionprovides an anti-human IL-21 monoclonal antibody comprising amino acidresidues 20 to 139 of SEQ ID NO: 61 and amino acid residues 23 to 129 ofSEQ ID NO: 69. In other embodiments, the present invention furthercomprises amino acid residues 1 to 139 of SEQ ID NO: 61 and amino acidresidues 1 to 129 of SEQ ID NO: 69. Another embodiment of the presentinvention provides a hybridoma designated 366.328.10.63, wherein thehybridoma is deposited with the American Type Culture Collection havingthe ATCC Patent Deposit Designation PTA-8789 (deposited Nov. 15, 2007,American Type Culture Collection, 10801 University Boulevard, Manassas,Va. 20110-2209), and the antibody produced by the hybridoma.

In another aspect, the present invention provides a hybridoma designated366.345.6.11, wherein the hybridoma is deposited with the American TypeCulture Collection having the ATCC Patent Deposit Designation PTA-8788and the includes the antibody produced by the hybridoma.

In another aspect of the present invention, the present inventionprovides an isolated monoclonal antibody that binds to a discontinuousepitope comprising at least two regions on an IL-21 protein, wherein thefirst region consists of at least one amino acid from residue Ile45 toresidue Leu56 of SEQ ID NO: 2 and the second region consists at leastone amino acid residue Glu129 to residue Leu144 of SEQ ID NO: 2. In oneembodiment, the invention provides that the first region consists ofbetween 1 and 12 amino acids from residue Ile45 to residue Leu56 of SEQID NO: 2, and the second region consists of between 1 and 16 amino acidsfrom residue Glu129 to residue Leu144 of SEQ ID NO: 2.

In a further aspect, the present invention provides a bispecific bindingcomposition that neutralizes both IL-21 and a second antigen related toautoimmune disease. Such bispecific binding compositions typicallycomprise (a) an isolated anti-IL-21 antibody and (b) an isolatedantibody to the second autoimmune disease-related antibody. In someembodiments, the anti-IL-21 antibody and the second antibody arecovalently linked via a linker. Particularly suitable linkers includepolypeptide linkers. In some variations, the anti-IL-21 antibody and thesecond antigen antibodies are single chain Fv fragments covalentlylinked to form a tandem single chain Fv (tascFv) or a bispecific singlechain Fv (biscFv). In some embodiments, the bispecific bindingcompositions further comprises a pharmaceutically acceptable carrier. Insome preferred variations, the bispecific antibody is a tascFv, abiscFv, or a biAb. In some embodiments, a bispecific antibody isPEGylated.

In each aspect of the inventions described above, included is anembodiment where the monoclonal antibody further comprises an Fcportion, and another embodiment, wherein the Fc portion is selected thegroup consisting of IgG1, IgG2 and IgG4 and another embodiment, whereinthe Fc portion has reduced effector function.

The second autoimmune disease-related antibody can be selected fromthose antigens who are believed to have an upregulating (oranti-suppressive) effect on B, T cells, or other immune cells, thushaving a supportive effect on autoimmune disease. Specific moleculescontemplated by the present invention for the second antibody include,but are not limited to, antibodies which specifically bind to ligandssuch as BLyS, APRIL, IL-6, TNFalpha, IL-15, IL-17F, IL-17A,cross-reactive antibodies to both IL-17F and -17A, IL-23p19, IL-17D, andIL-5. Other cytokine molecule antigens contemplated include IL-1, IL-18,IL-20, and IFNalpha (i.e., the production of antibodies that bindspecifically to these cytokines). The present invention alsocontemplates the production of antibodies that bind to the receptors forthese molecules where the antibody interferes with the ligand binding orsignaling function of the receptor, including but not limited to, IL-6R,IL-1R, TNFR, IL-17RA, IL-15R, IL-18R, IL-20RA, IFNa/bR, ICOS and LFA-1.

In another aspect, the present invention provides a method of treating Tfollicular helper cell-mediated or B cell-mediated diseases in a subjectby administering a therapeutic amount of the claimed anti-human IL-21monoclonal antibodies or a bispecific binding composition describedherein, wherein the T follicular helper cell-mediated and Bcell-mediated diseases are selected from the group consisting ofsystemic lupus erythematosus, autoimmune hearing loss, Graves' Disease,pemphigus vulgaris, myasthenia gravis, neuromyelitis optica,Goodpasture's syndrome, autoimmune nephritis, cryoglobulinemia, GuillainBane syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP),autoimmune hemolytic anemia, and idiopathic thrombocytopenic purpura(ITP).

In another aspect, the present invention provides a method of treatingTH1 cell-mediated or TH17 cell-mediated diseases in a subject byadministering a therapeutic amount of the claimed anti-human IL-21monoclonal antibodies bispecific binding composition described herein,wherein the TH1 cell-mediated or TH17 cell-mediated diseases areselected from the group consisting of psoriasis, spondyloarthropathy,reactive arthritis, enteropathic arthritis, autoimmune myocarditis,Kawasaki disease, celiac disease, uveitis, Behcet's disease, coronaryartery disease, chronic obstructive pulmonary disease (COPD), andinterstitial lung disease.

In another aspect, the present invention provides a method of treatinginflammatory bowel disease (IBD) in a subject by administering atherapeutic amount of the claimed anti-human IL-21 monoclonal antibodiesbispecific binding composition described herein, wherein theinflammatory bowel disease is selected from the group consisting ofCrohn's Disease, ulcerative colitis and irritable bowel syndrome.

In another aspect, the present invention provides a method of treatingrheumatoid arthritis in a subject by administering a therapeutic amountof the claimed anti-human IL-21 monoclonal antibodies or a bispecificbinding composition described herein.

In another aspect, the present invention provides a method of treatingmultiple sclerosis in a subject by administering a therapeutic amount ofthe claimed anti-human IL-21 monoclonal antibodies or a bispecificbinding composition described herein.

In another aspect, the present invention provides a method of treatingtype I diabetes (IDDM) in a subject by administering a therapeuticamount of the claimed anti-human IL-21 monoclonal antibodies or abispecific binding composition.

In another aspect, the present invention provides a method of treatingSjogren's syndrome in a subject by administering a therapeutic amount ofthe claimed anti-human IL-21 monoclonal antibodies or a bispecificbinding composition described herein.

In another aspect, the present invention provides a method of treating atransplant subject by administering a therapeutic amount of the claimedanti-human IL-21 monoclonal antibodies or a bispecific bindingcomposition described herein, wherein transplant rejection issuppressed, tolerance in the pre-transplant therapeutic regimen isestablished or alloantibody titers in the subject are reduced.

In another aspect, the present invention provides a method of treatingan autoimmune disease in a subject by administering a therapeutic amountof the claimed anti-human IL-21 monoclonal antibodies or a bispecificbinding composition described herein, wherein the autoimmune disease isselected from the group consisting of pancreatitis, inflammatory muscledisease (polymyositis, dermatomyositis), microscopic polyangiitis,autoimmune aplastic anemia, autoimmune thyroiditis, autoimmunehepatitis, Wegener's syndrome, diverticulosis, ankylosing spondylitis,scleroderma, systemic sclerosis, psoriatic arthritis, osteoarthritis,atopic dermatitis, vitiligo, graft vs. host disease (GVHD), cutaneous Tcell lymphoma (CTCL), glomerulonephritis, IgA nephropathy, highlysensitized transplant patients, anti-phospholipid syndrome, and asthma,and other autoimmune diseases, or other diseases mediated by IL-21 andIL-21 receptor agonists.

In another aspect, the present invention provides a method of treatingsystemic lupus erythematosus (SLE) in a subject by administering atherapeutic amount of the claimed anti-human IL-21 monoclonal antibodiesor a bispecific binding composition described herein.

In another aspect, the present invention provides a method of treatingpsoriasis in a subject by administering a therapeutic amount of theclaimed anti-human IL-21 monoclonal antibodies or a bispecific bindingcomposition described herein.

DESCRIPTION OF THE INVENTION

The following definitions are provided to facilitate understanding ofthe inventions described herein.

The terms “amino-terminal” and “carboxyl-terminal” are used herein todenote positions within polypeptides. Where the context allows, theseterms are used with reference to a particular sequence or portion of apolypeptide to denote proximity or relative position. For example, acertain sequence positioned carboxyl-terminal to a reference sequencewithin a polypeptide is located proximal to the carboxyl terminus of thereference sequence, but is not necessarily at the carboxyl terminus ofthe complete polypeptide.

The term “antagonist” refers to any compound including a protein,polypeptide, peptide, antibody, antibody fragment, large molecule, orsmall molecule (less than 10 kD), that decreases the activity,activation or function of another molecule. IL-21 antagonists cause atleast one of the following: decreased immune function of NK cells,dendritic cells, T cell subsets and B cell subsets; bind IL-21 such thatthe interaction of IL-21 protein with its receptor is blocked,inhibited, reduced or neutralized.

“Antibodies” (Abs) and “immunoglobulins” (Igs) are glycoproteins havingthe same structural characteristics. While antibodies exhibit bindingspecificity to a specific antigen, immunoglobulins include bothantibodies and other antibody-like molecules that lack antigenspecificity. Polypeptides of the latter kind are, for example, producedat low levels by the lymph system and at increased levels by myelomas.Thus, as used herein, the term “antibody” or “antibody peptide(s)”refers to an intact antibody, or a binding fragment thereof thatcompetes with the intact antibody for specific binding and includeschimeric, humanized, fully human, and bispecific antibodies. In certainembodiments, binding fragments are produced by recombinant DNAtechniques. In additional embodiments, binding fragments are produced byenzymatic or chemical cleavage of intact antibodies. Binding fragmentsinclude, but are not limited to, Fab, Fab′, F(ab′)₂, Fv, andsingle-chain antibodies, ScFv. “Native antibodies and immunoglobulins”are usually heterotetrameric glycoproteins of about 150,000 daltons,composed of two identical light (L) chains and two identical heavy (H)chains. Each light chain is linked to a heavy chain by one covalentdisulfide bond, while the number of disulfide-linkages varies betweenthe heavy chains of different immunoglobulin isotypes. Each heavy andlight chain also has regularly spaced intrachain disulfide bridges. Eachheavy chain has at one end a variable domain (VH) followed by a numberof constant domains. Each light chain has a variable domain at one end(VL) and a constant domain at its other end; the constant domain of thelight chain is aligned with the first constant domain of the heavychain, and the light chain variable domain is aligned with the variabledomain of the heavy chain. Particular amino acid residues are believedto form an interface between the light- and heavy-chain variable domains(Chothia et al., J. Mol. Biol. 186:651 (1985); Novotny and Haber, Proc.Natl. Acad. Sci. U.S.A. 82:4592 (1985)).

The term “chimeric antibody” or “chimeric antibodies” refers toantibodies whose light and heavy chain genes have been constructed,typically by genetic engineering, from immunoglobulin variable andconstant region genes belonging to different species. For example, thevariable segments of the genes from a mouse monoclonal antibody may bejoined to human constant segments, such as gamma 1 and gamma 3. Atypical therapeutic chimeric antibody is thus a hybrid protein composedof the variable or antigen-binding domain from a mouse antibody and theconstant domain from a human antibody, although other mammalian speciesmay be used. Specifically, a chimeric antibody is produced byrecombinant DNA technology in which all or part of the hinge andconstant regions of an immunoglobulin light chain, heavy chain, or both,have been substituted for the corresponding regions from anotheranimal's immunoglobulin light chain or heavy chain. In this way, theantigen-binding portion of the parent monoclonal antibody is graftedonto the backbone of another species' antibody.

The term “epitope” refers to any protein determinant capable of specificbinding to an immunoglobulin or T-cell receptor. Epitopic determinantsusually consist of chemically active surface groupings of molecules suchas amino acids or sugar side chains and usually have specific threedimensional structural characteristics, as well as specific chargecharacteristics. More specifically, the term “IL-21 epitope” as usedherein refers to a portion of the IL-21 polypeptide having antigenic orimmunogenic activity in an animal, preferably in a mammal, and mostpreferably in a mouse or a human. An epitope having immunogenic activityis a portion of a IL-21 polypeptide that elicits an antibody response inan animal. An epitope having antigenic activity is a portion of a IL-21polypeptide to which an antibody immuno specifically binds as determinedby any method well known in the art, for example, by immunoassays.Antigenic epitopes need not necessarily be immunogenic. “Discontinuousepitopes” are conformational epitopes formed from at least two separateregions in the primary sequence of the IL-21 protein. Conformationalepitopes lose the ability to specifically bind in the presence ofdenaturing solvents (e.g. in western blot analyses).

An “antigen-binding site of an antibody” is that portion of an antibodythat is sufficient to bind to its antigen. The minimum such region istypically a variable domain or a genetically engineered variant thereof.Single-domain binding sites can be generated from camelid antibodies(see Muyldermans and Lauwereys, J. Mol. Recog. 12:131-140, 1999; Nguyenet al., EMBO J. 19:921-930, 2000) or from V_(H) domains of other speciesto produce single-domain antibodies (“dAbs”; see Ward et al., Nature341:544-546, 1989; U.S. Pat. No. 6,248,516 to Winter et al.). In certainvariations, an antigen-binding site is a polypeptide region having only2 complementarity determining regions (CDRs) of a naturally ornon-naturally (e.g., mutagenized) occurring heavy chain variable domainor light chain variable domain, or combination thereof (see, e.g., Pessiet al., Nature 362:367-369, 1993; Qiu et al., Nature Biotechnol.25:921-929, 2007). More commonly, an antigen-binding site of an antibodycomprises both a heavy chain variable domain and a light chain variabledomain that bind to a common epitope. Within the present invention, amolecule that “comprises an antigen-binding site of an antibody” mayfurther comprise one or more of a second antigen-binding site of anantibody (which may bind to the same or a different epitope or to thesame or a different antigen), a peptide linker, an immunoglobulinconstant domain, an immunoglobulin hinge, an amphipathic helix (see Packand Pluckthun, Biochem. 31:1579-1584, 1992), a non-peptide linker, anoligonucleotide (see Chaudri et al., FEBS Letters 450:23-26, 1999), andthe like, and may be a monomeric or multimeric protein. Examples ofmolecules comprising an antigen-binding site of an antibody are known inthe art and include, for example, Fv fragments, single-chain Fvfragments (scFv), Fab fragments, diabodies, minibodies, Fab-scFvfusions, bispecific (scFv)₄-IgG, and bispecific (scFv)₂-Fab. (See, e.g.,Hu et al., Cancer Res. 56:3055-3061, 1996; Atwell et al., MolecularImmunology 33:1301-1312, 1996; Carter and Merchant, Curr. Opin.Biotechnol. 8:449-454, 1997; Zuo et al., Protein Engineering 13:361-367,2000; and Lu et al., J. Immunol. Methods 267:213-226, 2002.)

As used herein, the term “immunoglobulin” refers to a protein consistingof one or more polypeptides substantially encoded by immunoglobulingene(s). One form of immunoglobulin constitutes the basic structuralunit of an antibody. This form is a tetramer and consists of twoidentical pairs of immunoglobulin chains, each pair having one light andone heavy chain. In each pair, the light and heavy chain variableregions are together responsible for binding to an antigen, and theconstant regions are responsible for the antibody effector functions.Immunoglobulins typically function as antibodies in a vertebrateorganism. Five classes of immunoglobulin protein (IgG, IgA, IgM, IgD,and IgE) have been identified in higher vertebrates. IgG comprises themajor class; it normally exists as the second most abundant proteinfound in plasma. In humans, IgG consists of four subclasses, designatedIgG1, IgG2, IgG3, and IgG4. The heavy chain constant regions of the IgGclass are identified with the Greek symbol γ. For example,immunoglobulins of the IgG1 subclass contain a γ1 heavy chain constantregion. Each immunoglobulin heavy chain possesses a constant region thatconsists of constant region protein domains (C_(H)1, hinge, C_(H)2, andC_(H)3; IgG3 also contains a C_(H)4 domain) that are essentiallyinvariant for a given subclass in a species. DNA sequences encodinghuman and non-human immunoglobulin chains are known in the art. (See,e.g., Ellison et al., DNA 1:11-18, 1981; Ellison et al., Nucleic AcidsRes. 10:4071-4079, 1982; Kenten et al., Proc. Natl. Acad. Sci. USA79:6661-6665, 1982; Seno et al., Nuc. Acids Res. 11:719-726, 1983;Riechmann et al., Nature 332:323-327, 1988; Amster et al., Nuc. AcidsRes. 8:2055-2065, 1980; Rusconi and Kohler, Nature 314:330-334, 1985;Boss et al., Nuc. Acids Res. 12:3791-3806, 1984; Bothwell et al., Nature298:380-382, 1982; van der Loo et al., Immunogenetics 42:333-341, 1995;Karlin et al., J. Mol. Evol. 22:195-208, 1985; Kindsvogel et al., DNA1:335-343, 1982; Breiner et al., Gene 18:165-174, 1982; Kondo et al.,Eur. J. Immunol. 23:245-249, 1993; and GenBank Accession No. J00228.)For a review of immunoglobulin structure and function see Putnam, ThePlasma Proteins, Vol V, Academic Press, Inc., 49-140, 1987; and Padlan,Mol. Immunol. 31:169-217, 1994. The term “immunoglobulin” is used hereinfor its common meaning, denoting an intact antibody, its componentchains, or fragments of chains, depending on the context.

Full-length immunoglobulin “light chains” (about 25 Kd or 214 aminoacids) are encoded by a variable region gene at the NH₂-terminus(encoding about 110 amino acids) and a by a kappa or lambda constantregion gene at the COOH-terminus. Full-length immunoglobulin “heavychains” (about 50 Kd or 446 amino acids) are encoded by a variableregion gene (encoding about 116 amino acids) and a gamma, mu, alpha,delta, or epsilon constant region gene (encoding about 330 amino acids),the latter defining the antibody's isotype as IgG, IgM, IgA, IgD, orIgE, respectively. Within light and heavy chains, the variable andconstant regions are joined by a “J” region of about 12 or more aminoacids, with the heavy chain also including a “D” region of about 10 moreamino acids. (See generally Fundamental Immunology (Paul, ed., RavenPress, N.Y., 2nd ed. 1989), Ch. 7).

An immunoglobulin “Fv” fragment contains a heavy chain variable domain(V_(H)) and a light chain variable domain (V_(L)), which are heldtogether by non-covalent interactions. An immunoglobulin Fv fragmentthus contains a single antigen-binding site. The dimeric structure of anFv fragment can be further stabilized by the introduction of a disulfidebond via mutagenesis. (See Almog et al., Proteins 31:128-138, 1998.)

As used herein, the terms “single-chain Fv” and “single-chain antibody”refer to antibody fragments that comprise, within a single polypeptidechain, the variable regions from both heavy and light chains, but lackconstant regions. In general, a single-chain antibody further comprisesa polypeptide linker between the V_(H) and V_(L) domains, which enablesit to form the desired structure that allows for antigen binding.Single-chain antibodies are discussed in detail by, for example,Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113(Rosenburg and Moore eds., Springer-Verlag, New York, 1994), pp.269-315. (See also WIPO Publication WO 88/01649; U.S. Pat. Nos.4,946,778 and 5,260,203; Bird et al., Science 242:423-426, 1988.)Single-chain antibodies can also be bi-specific and/or humanized.

A “Fab fragment” contains one light chain and the C_(H)1 and variableregions of one heavy chain. The heavy chain of a Fab fragment cannotform a disulfide bond with another heavy chain molecule.

A “Fab′ fragment” contains one light chain and one heavy chain thatcontains more of the constant region, between the C_(H)1 and C_(H)2domains, such that an interchain disulfide bond can be formed betweentwo heavy chains to form a F(ab′)₂ molecule.

A “F(ab′)₂ fragment” contains two light chains and two heavy chainscontaining a portion of the constant region between the C_(H)1 andC_(H)2 domains, such that an interchain disulfide bond is formed betweentwo heavy chains.

An immunoglobulin “Fc fragment” (or Fc domain) is the portion of anantibody that is responsible for binding to antibody receptors on cellsand the C1q component of complement. Fc stands for “fragmentcrystalline,” the fragment of an antibody that will readily form aprotein crystal. Distinct protein fragments, which were originallydescribed by proteolytic digestion, can define the overall generalstructure of an immunoglobulin protein. As originally defined in theliterature, the Fc fragment consists of the disulfide-linked heavy chainhinge regions, C_(H)2, and C_(H)3 domains. However, more recently theterm has been applied to a single chain consisting of C_(H)3, C_(H)2,and at least a portion of the hinge sufficient to form adisulfide-linked dimer with a second such chain. For a review ofimmunoglobulin structure and function, see Putnam, The Plasma Proteins,Vol. V (Academic Press, Inc., 1987), pp. 49-140; and Padlan, Mol.Immunol. 31:169-217, 1994. As used herein, the term Fc includes variantsof naturally occurring sequences.

An immunoglobulin light or heavy chain variable region consists of a“framework” region interrupted by three hypervariable regions. Thus, theterm “hypervariable region” refers to the amino acid residues of anantibody that are responsible for antigen binding. The hypervariableregion comprises amino acid residues from a “Complementarity DeterminingRegion” or “CDR” (e.g., in human, residues 24-34 (L1), 50-56 (L2), and89-97 (L3) in the light chain variable domain and residues 31-35 (H1),50-65 (H2) and 95-102 (H3) in the heavy chain variable domain (aminoacid sequence numbers based on the EU index; see Kabat et al., Sequencesof Proteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)) and/or thoseresidues from a “hypervariable loop” (in human, residues 26-32 (L1),50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32(H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain;Chothia and Lesk, J. Mol. Biol. 196: 901-917, 1987) (both of which areincorporated herein by reference). “Framework Region” or “FR” residuesare those variable domain residues other than the hypervariable regionresidues as herein defined. The sequences of the framework regions ofdifferent light or heavy chains are relatively conserved within aspecies. Thus, a “human framework region” is a framework region that issubstantially identical (about 85% or more, usually 90-95% or more) tothe framework region of a naturally occurring human immunoglobulin. Theframework region of an antibody, that is the combined framework regionsof the constituent light and heavy chains, serves to position and alignthe CDR's. The CDR's are primarily responsible for binding to an epitopeof an antigen. CDRs L1, L2, and L3 of the V_(L) domain are also referredto herein, respectively, as LCDR1, LCDR2, and LCDR3; CDRs H1, H2, and H3of the V_(H) domain are also referred to herein, respectively, as HCDR1,HCDR2, and HCDR3.

As used herein, the term “human antibody” includes an antibody that hasan amino acid sequence of a human immunoglobulin and includes antibodiesisolated from human immunoglobulin libraries or from animals transgenicfor one or more human immunoglobulin genes and that do not expressendogenous immunoglobulins, as described, for example, in U.S. Pat. No.5,939,598 to Kucherlapati et al.

The term “humanized immunoglobulin” refers to an immunoglobulincomprising a human framework region and one or more CDR's from anon-human (e.g., a mouse or rat) immunoglobulin. The non-humanimmunoglobulin providing the CDR's is called the “donor” and the humanimmunoglobulin providing the framework is called the “acceptor.”Constant regions need not be present, but if they are, they must besubstantially identical to human immunoglobulin constant regions, i.e.,at least about 85-90%, preferably about 95% or more identical. Hence,all parts of a humanized immunoglobulin, except possibly the CDR's, aresubstantially identical to corresponding parts of natural humanimmunoglobulin sequences. In some instances, humanized antibodies mayretain non-human residues within the human variable region frameworkdomains to enhance proper binding characteristics (e.g., mutations inthe frameworks may be required to preserve binding affinity when anantibody is humanized). A “humanized antibody” is an antibody comprisinga humanized light chain and a humanized heavy chain immunoglobulin. Forexample, a humanized antibody would not encompass a typical chimericantibody as defined above because, e.g., the entire variable region of achimeric antibody is non-human.

A “bispecific” or “bifunctional” antibody is a hybrid antibody havingtwo different heavy/light chain pairs and two different binding sites.Bispecific antibodies may be produced by a variety of methods including,but not limited to, fusion of hybridomas or linking of Fab′ fragments.See, e.g., Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321, 1990;Kostelny et al., J. Immunol. 148:1547-1553, 1992.

A “bivalent antibody” other than a “multispecific” or “multifunctional”antibody, in certain embodiments, is an antibody comprising two bindingsites having identical antigenic specificity.

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy chain variabledomain (V_(H)) connected to a light chain variable domain (V_(L)) in thesame polypeptide chain (V_(H)-V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, for example, EP 404,097; WO 93/11161; and Hollinger et al.,Proc. Natl. Acad. Sci. USA 90:6444-6448, 1993.

The term “minibody” refers herein to a polypeptide that encodes only 2complementarity determining regions (CDRs) of a naturally ornon-naturally (e.g., mutagenized) occurring heavy chain variable domainor light chain variable domain, or combination thereof. Examples ofminibodies are described by, e.g., Pessi et al., Nature 362:367-369,1993; and Qiu et al., Nature Biotechnol. 25:921-929, 2007.

The term “linear antibodies” refers to the antibodies described inZapata et al., Protein Eng. 8:1057-1062, 1995. Briefly, these antibodiescomprise a pair of tandem Fd segments (V_(H)-C_(H1)-V_(H)-C_(H1)) whichform a pair of antigen binding regions. Linear antibodies can bebispecific or monospecific.

The term “monoclonal antibody” as used herein is not limited toantibodies produced through hybridoma technology. The term “monoclonalantibody” refers to an antibody that is derived from a single clone,including any eukaryotic, prokaryotic, or phage clone, and not themethod by which it is produced.

The term “parent antibody” as used herein refers to an antibody which isencoded by an amino acid sequence used for the preparation of thevariant. Preferably, the parent antibody has a human framework regionand, if present, has human antibody constant region(s). For example, theparent antibody may be a humanized or human antibody.

A “variant” anti-IL-21 or antibody to a second antigen related toautoimmune disease, refers herein to a molecule which differs in aminoacid sequence from a “parent” antibody amino acid sequence by virtue ofaddition, deletion and/or substitution of one or more amino acidresidue(s) in the parent antibody sequence. In the preferred embodiment,the variant comprises one or more amino acid substitution(s) in one ormore hypervariable region(s) of the parent antibody. For example, thevariant may comprise at least one, e.g., from about one to about ten,and preferably from about two to about five, substitutions in one ormore hypervariable regions of the parent antibody. Ordinarily, thevariant will have an amino acid sequence having at least 75% amino acidsequence identity with the parent antibody heavy or light chain variabledomain sequences, more preferably at least 80%, more preferably at least85%, more preferably at least 90%, and most preferably at least 95%.Identity or homology with respect to this sequence is defined herein asthe percentage of amino acid residues in the candidate sequence that areidentical with the parent antibody residues, after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity. None of N-terminal, C-terminal, or internalextensions, deletions, or insertions into the antibody sequence shall beconstrued as affecting sequence identity or homology. The variantretains the ability to bind the target antigen and preferably hasproperties which are superior to those of the parent antibody. Forexample, the variant may have a stronger binding affinity, enhancedability to inhibit antigen biological activity. To analyze suchproperties, one should compare a Fab form of the variant to a Fab formof the parent antibody or a full length form of the variant to a fulllength form of the parent antibody, for example, since it has been foundthat the format of an antibody impacts its activity in the biologicalactivity assays disclosed herein. The variant antibody of particularinterest herein is one which displays about at least a 3 fold, 5 fold,10 fold, 20 fold, or 50 fold, enhancement in biological activity whencompared to the parent antibody.

Two amino acid sequences have “100% amino acid sequence identity” if theamino acid residues of the two amino acid sequences are the same whenaligned for maximal correspondence. Similarly, two nucleotide sequenceshave “100% nucleotide sequence identity” if the nucleotide residues ofthe two nucleotide sequences are the same when aligned for maximalcorrespondence. Sequence comparisons can be performed using standardsoftware programs such as those included in the LASERGENE bioinformaticscomputing suite, which is produced by DNASTAR (Madison, Wis.). Othermethods for comparing two nucleotide or amino acid sequences bydetermining optimal alignment are well-known to those of skill in theart. (See, e.g., Peruski and Peruski, The Internet and the New Biology:Tools for Genomic and Molecular Research (ASM Press, Inc. 1997); Wu etal. (eds.), “Information Superhighway and Computer Databases of NucleicAcids and Proteins,” in Methods in Gene Biotechnology 123-151 (CRCPress, Inc. 1997); Bishop (ed.), Guide to Human Genome Computing (2nded., Academic Press, Inc. 1998).) Two nucleotide or amino acid sequencesare considered to have “substantially similar sequence identity” or“substantial sequence identity” if the two sequences have at least 80%,at least 90%, or at least 95% sequence identity relative to each other.

Percent sequence identity is determined by conventional methods. See,e.g., Altschul et al., Bull. Math. Bio. 48:603, 1986, and Henikoff andHenikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1992. For example, twoamino acid sequences can be aligned to optimize the alignment scoresusing a gap opening penalty of 10, a gap extension penalty of 1, and the“BLOSUM62” scoring matrix of Henikoff and Henikoff, supra, as shown inTable 1 (amino acids are indicated by the standard one-letter codes).The percent identity is then calculated as: ([Total number of identicalmatches]/[length of the longer sequence plus the number of gapsintroduced into the longer sequence in order to align the twosequences])(100).

TABLE 1 BLOSUM62 Scoring Matrix A R N D C Q E G H I L K M F P S T W Y VA 4 R −1 5 N −2 0 6 D −2 −2 1 6 C 0 −3 −3 −3 9 Q −1 1 0 0 −3 5 E −1 0 02 −4 2 5 G 0 −2 0 −1 −3 −2 −2 6 H −2 0 1 −1 −3 0 0 −2 8 I −1 −3 −3 −3 −1−3 −3 −4 −3 4 L −1 −2 −3 −4 −1 −2 −3 −4 −3 2 4 K −1 2 0 −1 −3 1 1 −2 −1−3 −2 5 M −1 −1 −2 −3 −1 0 −2 −3 −2 1 2 −1 5 F −2 −3 −3 −3 −2 −3 −3 −3−1 0 0 −3 0 6 P −1 −2 −2 −1 −3 −1 −1 −2 −2 −3 −3 −1 −2 −4 7 S 1 −1 1 0−1 0 0 0 −1 −2 −2 0 −1 −2 −1 4 T 0 −1 0 −1 −1 −1 −1 −2 −2 −1 −1 −1 −1 −2−1 1 5 W −3 −3 −4 −4 −2 −2 −3 −2 −2 −3 −2 −3 −1 1 −4 −3 −2 11 Y −2 −2 −2−3 −2 −1 −2 −3 2 −1 −1 −2 −1 3 −3 −2 −2 2 7 V 0 −3 −3 −3 −1 −2 −2 −3 −33 1 −2 1 −1 −2 −2 0 −3 −1 4Those skilled in the art appreciate that there are many establishedalgorithms available to align two amino acid sequences. The “FASTA”similarity search algorithm of Pearson and Lipman is a suitable proteinalignment method for examining the level of identity shared by an aminoacid sequence disclosed herein and a second amino acid sequence. TheFASTA algorithm is described by Pearson and Lipman, Proc. Nat'l Acad.Sci. USA 85:2444, 1988, and by Pearson, Meth. Enzymol. 183:63, 1990.Briefly, FASTA first characterizes sequence similarity by identifyingregions shared by the query sequence and a test sequence that haveeither the highest density of identities (if the ktup variable is 1) orpairs of identities (if ktup=2), without considering conservative aminoacid substitutions, insertions, or deletions. The ten regions with thehighest density of identities are then rescored by comparing thesimilarity of all paired amino acids using an amino acid substitutionmatrix, and the ends of the regions are “trimmed” to include only thoseresidues that contribute to the highest score. If there are severalregions with scores greater than the “cutoff” value (calculated by apredetermined formula based upon the length of the sequence and the ktupvalue), then the trimmed initial regions are examined to determinewhether the regions can be joined to form an approximate alignment withgaps. Finally, the highest scoring regions of the two amino acidsequences are aligned using a modification of theNeedleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol. Biol.48:444, 1970; Sellers, SIAM J. Appl. Math. 26:787, 1974), which allowsfor amino acid insertions and deletions. Illustrative parameters forFASTA analysis are: ktup=1, gap opening penalty=10, gap extensionpenalty=1, and substitution matrix=BLOSUM62. These parameters can beintroduced into a FASTA program by modifying the scoring matrix file(“SMATRIX”), as explained in Appendix 2 of Pearson, Meth. Enzymol.183:63, 1990.

FASTA can also be used to determine the sequence identity of nucleicacid molecules using a ratio as disclosed above. For nucleotide sequencecomparisons, the ktup value can range between one to six, preferablyfrom three to six, most preferably three, with other parameters set asdescribed above.

FASTA can also be used to determine the sequence identity of nucleicacid molecules using a ratio as disclosed above. For nucleotide sequencecomparisons, the ktup value can range between one to six, preferablyfrom three to six, most preferably three, with other parameters set asdescribed above.

Other than percent homology, variant antibodies can also be describedthe number of amino acid changes from the sequences which are disclosedherein. For example, for full length heavy or light variable chains, thepresention invention contemplates 20 or fewer conservative amino acidsubstitutions from the amino acids described. Additionally, CDRs canvary from the sequences disclosed therein depending on the particularCDR at issue. For example, CDR-1 from either a light or heavy chain canvery by four or fewer amino acid substitutions. CDR-2, from either alight or heavy chain variable region can vary by two or fewer amino acidsubstitutions. CDR-3, from either a light or heavy chain variable regioncan vary by four or fewer amino acid substitutions. Finally, theframework portion of the light or heavy chains can vary by five or feweramino acid substitutions and remain within the presently contemplatedinvention. It should be noted that antibodies that comprises such aminoacid changes would retain their ability to bind to the designatedantigen, i.e., IL-21 or the second antigen involved in autoimmunedisease.

The term “synergistic” is used herein to denote a biological or clinicalactivity of two or more therapeutic agents that when measured is atleast greater than either agent alone.

The presently described antibodies are named according to the followingnumeric convention: a three digit fusion number, a two digit master wellnumber, followed by one or more single or double digit cloning rounddesignations. Each of these numbers are separated by a period. Thus,antibody “378.78.1” indicates it is from fusion 378, mater well 78,cloning round 1. It is anticipated that antibodies derived from the samefusion and master well but various cloning rounds will be essentiallyidentical to each other. Thus, antibody 378.78.1 and antibody378.78.1.44, although from different cloning rounds, would be expectedto be the same molecule.

The present invention provides monoclonal antibodies and antibodyfragments that specifically bind to IL-21 proteins and polypeptides.Human and mouse IL-21 polypeptides, proteins and polynucleotidesencoding the polypeptides are disclosed in Parrish-Novak et al., Nature408:57-63, 2003; U.S. Pat. Nos. 6,307,024 and 6,686,178; and 7,250,274.Described herein are structural and functional characteristics definingregions (epitopes) of the human IL-21 protein that have been identifiedas targets for a therapeutic monoclonal antibody. Exemplary humananti-human IL-21 monoclonal antibodies are presented. Certain of theseantibodies have the ability to bind native human IL-21, recombinantwildtype human IL-21, a recombinant mutant IL-21 protein and/or peptideregions of human IL-21.

The present invention provides anti-IL-21 antibodies which are useful intherapeutic treatment of autoimmune and inflammatory diseases. Forexample, anti-IL-21 antibodies are useful in the treatment of psoriasis,pancreatitis, type I diabetes (IDDM), Graves' Disease, inflammatorybowel disease (IBD), Crohn's Disease, ulcerative colitis, irritablebowel syndrome, multiple sclerosis, rheumatoid arthritis, reactivearthritis, enteropathic arthritis, spondyloarthropathy, autoimmunemyocarditis, Kawasaki disease, celiac disease, uveitis, Behcet'sdisease, coronary artery disease, chronic obstructive pulmonary disease(COPD), interstitial lung disease, inflammatory muscle disease(polymyositis, dermatomyositis), microscopic polyangiitis, autoimmuneaplastic anemia, autoimmune thyroiditis, autoimmune hepatitis, Wegener'ssyndrome, diverticulosis, systemic lupus erythematosus, ankylosingspondylitis, scleroderma, systemic sclerosis, psoriatic arthritis,osteoarthritis, atopic dermatitis, vitiligo, graft vs. host disease(GVHD), cutaneous T cell lymphoma (CTCL), Sjogren's syndrome,glomerulonephritis, IgA nephropathy, autoimmune nephritis, pemphigusvulgaris, myasthenia gravis, autoimmune hearing loss, neuromyelitisoptica, Goodpasture's syndrome, cryoglobulinemia, Guillain Barresyndrome, chronic inflammatory demyelinating polyneuropathy (CIDP),autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura (ITP),transplant rejection, highly sensitized transplant patients,anti-phospholipid syndrome, allergy, and asthma, and other autoimmunediseases, or other diseases mediated by IL-21 and IL-21 receptoragonists.

Five classes of immunoglobulin, IgG, IgA, IgM, IgD, and IgE, have beenidentified in higher vertebrates. IgG, IgD, and IgE proteins arecharacteristically disulfide linked heterotetramers consisting of twoidentical heavy chains and two identical light chains. Typically, IgM isfound as a pentamer of a tetramer, whereas IgA occurs as a dimer of atetramer. Modifications can be introduced in the immunoglobulin moiety.

IgG comprises the major class as it normally exists as the second mostabundant protein found in plasma. In humans, IgG consists of foursubclasses, designated IgG1, IgG2, IgG3, and IgG4. Each immunoglobulinheavy chain possesses a constant region that consists of constant regionprotein domains (C_(H)1, hinge, C_(H)2, and C_(H)3) that are invariantfor a given subclass. The heavy chain constant regions of the IgG classare identified with the Greek symbol γ. For example, immunoglobulins ofthe IgG1 subclass contain a γ1 heavy chain constant region.

The Fc fragment, or Fc domain, consists of the disulfide linked heavychain hinge regions, C_(H)2, and C_(H)3 domains. In immunoglobulinfusion proteins, Fc domains of the IgG1 subclass are often used as theimmunoglobulin moiety, because IgG1 has the longest serum half-life ofany of the serum proteins. Lengthy serum half-life can be a desirableprotein characteristic for animal studies and potential humantherapeutic use. In addition, the IgG1 subclass possesses the strongestability to carry out antibody mediated effector functions. The primaryeffector function that may be most useful in an immunoglobulin fusionprotein is the ability for an IgG1 antibody to mediate antibodydependent cellular cytotoxicity. On the other hand, this could be anundesirable function for a fusion protein that functions primarily as anantagonist. Several of the specific amino acid residues that areimportant for antibody constant region-mediated activity in the IgG1subclass have been identified. Inclusion or exclusion of these specificamino acids therefore allows for inclusion or exclusion of specificimmunoglobulin constant region-mediated activity (see, U.S. Pat. Nos.5,648,260; 5,624,821).

Modified human IgG1 Fc have been generated for creating Fc fusionproteins. For example, Fc4, Fc5, and Fc6 mutations to reduce effectorfunctions mediated by the Fc by reducing FcγRI binding and complementC1q binding are described in U.S. Patent Application 2006-0034852,incorporated by reference herein in its entirety. Specifically, threeamino acid substitutions were introduced to reduce FcγRI binding. Theseare the substitutions at EU index positions 234, 235, and 237.Substitutions at these positions have been shown to reduce binding toFcγRI (Duncan et al., Nature 332:563 (1988)). These amino acidsubstitutions may also reduce FcγRIIa binding, as well as FcγRIIIbinding (Sondermann et al., Nature 406:267 (2000); Wines et al., J.Immunol. 164:5313 (2000)). These mutations do not alter binding to FcRn,which promotes long serum half-life by salvaging IgG through anendocytic recycling pathway.

Several groups have described the relevance of EU index positions 330and 331 in complement C1q binding and subsequent complement fixation(Canfield and Morrison, J. Exp. Med. 173:1483 (1991); Tao et al., J.Exp. Med. 178:661 (1993)). Amino acid substitutions at these positionswere introduced in Fc4 to reduce complement fixation. The C_(H)3 domainof Fc4 is identical to that found in the corresponding wild-typepolypeptide, except for the stop codon, which was changed from TGA toTAA to eliminate a potential dam methylation site when the cloned DNA isgrown in dam plus strains of E. coli. In Fc5, the arginine residue at EUindex position 218 is a lysine and the remainder of the Fc5 sequencematches the above description for Fc4.

The present invention also includes genetically altered antibodies thatare functionally equivalent to the above-described antibodies. Modifiedantibodies providing improved stability and/or therapeutic efficacy arepreferred. Examples of modified antibodies include those withconservative substitutions of amino acid residues, and one or moredeletions or additions of amino acids which do not significantlydeleteriously alter the antigen binding utility. Substitutions can rangefrom changing or modifying one or more amino acid residues to completeredesign of a region as long as the therapeutic utility is maintained.Antibodies of the present invention can be can be modifiedpost-translationally (e.g., acetylation, and phosphorylation) or can bemodified synthetically (e.g., the attachment of a labeling group).

In certain embodiments, a bispecific binding composition of theinvention neutralizes both IL-21 and a biological activity of a secondtarget molecule, generally an antigen associated with autoimmunedisease, and comprises an anti-IL-21 antibody as described herein.Accordingly, in particular variations, a bispecific binding compositioncomprises an anti-IL-21 antibody as described herein and a secondbinding entity capable of neutralizing the activity of the secondantigen.

In certain embodiments, two or more different entities of a bispecificbinding composition are linked via linker to form a multimer (e.g., adimer). For example, in the case of a bispecific binding compositioncomprising a fusion of at least two polypeptide components (e.g., ananti-IL-21 antibody and another polypeptide component), a peptide linkersequence may be employed to separate, for example, the polypeptidecomponents by a distance sufficient to ensure that each polypeptidefolds into its secondary and tertiary structures. Fusion proteins maygenerally be prepared using standard techniques, including chemicalconjugation. Fusion proteins can also be expressed as recombinantproteins in an expression system by standard techniques. Suitablelinkers are further described herein, infra.

A linker can be naturally-occurring, synthetic, or a combination ofboth. For example, a synthetic linker can be a randomized linker, e.g.,both in sequence and size. In one aspect, the randomized linker cancomprise a fully randomized sequence, or optionally, the randomizedlinker can be based on natural linker sequences. The linker cancomprise, for example, a non-polypeptide moiety (e.g., apolynucleotide), a polypeptide, or the like.

A linker can be rigid, or alternatively, flexible, or a combination ofboth. Linker flexibility can be a function of the composition of boththe linker and the subunits that the linker interacts with. The linkerjoins two selected binding entities (e.g., two separate polypeptides orproteins, such as two different antibodies) and maintains the entitiesas separate and discrete. The linker can allow the separate, discretedomains to cooperate yet maintain separate properties such as multipleseparate binding sites for the same target in a multimer or, forexample, multiple separate binding sites for different targets in amultimer. In some cases, a disulfide bridge exists between two linkedbinding entities or between a linker and a binding entity.

Choosing a suitable linker for a specific case where two or more bindingentities are to be connected may depend on a variety of parametersincluding, e.g., the nature of the binding entities, the structure andnature of the target to which the bispecific composition should bind,and/or the stability of the linker (e.g., peptide linker) towardsproteolysis and oxidation.

Particularly suitable linker polypeptides predominantly include aminoacid residues selected from Glycine (Gly), Serine (Ser), Alanine (Ala),and Threonine (Thr). For example, the peptide linker may contain atleast 75% (calculated on the basis of the total number of residuespresent in the peptide linker), such as at least 80%, at least 85%, orat least 90% of amino acid residues selected from Gly, Ser, Ala, andThr. The peptide linker may also consist of Gly, Ser, Ala and/or Thrresidues only. The linker polypeptide should have a length that isadequate to link two binding entities in such a way that they assume thecorrect conformation relative to one another so that they retain thedesired activity, such as binding to a target molecule as well as otheractivities that may be associated with such target binding (e.g.,agonistic or antagonistic activity for a given biomolecule).

A suitable length for this purpose is, e.g., a length of at least oneand typically fewer than about 50 amino acid residues, such as 2-25amino acid residues, 5-20 amino acid residues, 5-15 amino acid residues,8-12 amino acid residues or 11 residues. Other suitable polypeptidelinker sizes may include, e.g., from about 2 to about 15 amino acids,from about 3 to about 15, from about 4 to about 12, about 10, about 8,or about 6 amino acids. The amino acid residues selected for inclusionin the linker polypeptide should exhibit properties that do notinterfere significantly with the activity or function of the polypeptidemultimer. Thus, the peptide linker should, on the whole, not exhibit acharge that would be inconsistent with the activity or function of themultimer, or interfere with internal folding, or form bonds or otherinteractions with amino acid residues in one or more of the domains thatwould seriously impede the binding of the multimer to the target inquestion.

The use of naturally occurring as well as artificial peptide linkers toconnect polypeptides into novel linked fusion polypeptides is well-knownin the art. (See, e.g., Hallewell et al., J. Biol. Chem. 264, 5260-5268,1989; Alfthan et al., Protein Eng. 8, 725-731, 1995; Robinson and Sauer,Biochemistry 35, 109-116, 1996; Khandekar et al., J. Biol. Chem. 272,32190-32197, 1997; Fares et al., Endocrinology 139, 2459-2464, 1998;Smallshaw et al., Protein Eng. 12, 623-630, 1999; U.S. Pat. No.5,856,456.)

One example where the use of peptide linkers is widespread is forproduction of single-chain antibodies where the variable regions of alight chain (V_(L)) and a heavy chain (V_(H)) are joined through anartificial linker, and a large number of publications exist within thisparticular field (see, for example, the linkers described in Le Gall etal., Protein Eng. Des. Sel., 17(4): 357-66, 2004 and Volkel et al.,Protein Eng., 14(10): 815-23, 2001). Other linkers have been used, andphage display technology, as well as selective infective phagetechnology, has been used to diversify and select appropriate linkersequences (Tang et al., J. Biol. Chem. 271, 15682-15686, 1996; Henneckeet al., Protein Eng. 11, 405-410, 1998). Peptide linkers have been usedto connect individual chains in hetero- and homo-dimeric proteins suchas the T-cell receptor, the lambda Cro repressor, the P22 phage Arcrepressor, IL-12, TSH, FSH, IL-5, and interferon-γ. Peptide linkers havealso been used to create fusion polypeptides. Various linkers have beenused, and, in the case of the Arc repressor, phage display has been usedto optimize the linker length and composition for increased stability ofthe single-chain protein (see Robinson and Sauer, Proc. Natl. Acad. Sci.USA 95, 5929-5934, 1998).

Still another way of obtaining a suitable linker is by optimizing asimple linker through random mutagenesis.

As discussed above, it is generally preferred that the peptide linkerpossess at least some flexibility. Accordingly, in some variations, thepeptide linker contains 1-25 glycine residues, 5-20 glycine residues,5-15 glycine residues, or 8-12 glycine residues. Particularly suitablepeptide linkers typically contain at least 50% glycine residues, such asat least 75% glycine residues. In some embodiments, a peptide linkercomprises glycine residues only. In certain variations, the peptidelinker comprises other residues in addition to the glycine. Preferredresidues in addition to glycine include Ser, Ala, and Thr, particularlySer.

In some cases, it may be desirable or necessary to provide some rigidityinto the peptide linker. This may be accomplished by including prolineresidues in the amino acid sequence of the peptide linker. Thus, inanother embodiment, a peptide linker comprises at least one prolineresidue in the amino acid sequence of the peptide linker. For example, apeptide linker can have an amino acid sequence wherein at leak 25%(e.g., at least 50% or at least 75%) of the amino acid residues areproline residues. In one particular embodiment of the invention, thepeptide linker comprises proline residues only.

In some embodiments, a peptide linker is modified in such a way that anamino acid residue comprising an attachment group for a non-polypeptidemoiety is introduced. Examples of such amino acid residues may be acysteine or a lysine residue (to which the non-polypeptide moiety isthen subsequently attached). Another alternative is to include an aminoacid sequence having an in vivo N-glycosylation site (thereby attachinga sugar moiety (in vivo) to the peptide linker). An additional option isto genetically incorporate non-natural amino acids using evolved tRNAsand tRNA synthetases (see, e.g., U.S. Patent Application Publication2003/0082575) into a polypeptide binding entity or peptide linker. Forexample, insertion of keto-tyrosine allows for site-specific coupling toan expressed polypeptide.

In certain variations, a peptide linker comprises at least one cysteineresidue, such as one cysteine residue. For example, in some embodiments,a peptide linker comprises at least one cysteine residue and amino acidresidues selected from the group consisting of Gly, Ser, Ala, and Thr.In some such embodiments, a peptide linker comprises glycine residuesand cysteine residues, such as glycine residues and cysteine residuesonly. Typically, only one cysteine residue will be included per peptidelinker.

As previously noted, in certain embodiments, a bispecific bindingcomposition comprises an anti-IL-21 antibody and an antibody specificfor a second antigen. In some such embodiments, the anti-IL-21 andantibodies to the second antigen are covalently linked (e.g., via apeptide linker) to form a bispecific antibody. In some variations, thebispecific antibody comprises an immunoglobulin heavy chain constantregion such as, for example, an Fc fragment. Particularly suitable Fcfragments include, for example, Fc fragments comprising an Fc regionmodified to reduce or eliminate one or more effector functions.

In certain preferred embodiments, a bispecific antibody in accordancewith the present invention is a tandem single chain Fv (tascFv),bispecific single chain Fv (biscFv), or a bispecific antibody (biAb).For the tascFv molecule, two scFv molecules are constructed such thatone scFv is amino terminal to the other one in a tandem configuration.This can be done in each orientation. Tandem scFv molecules can beprepared with a linker between the scFv entities. In some embodiments,the linker is a Gly-Ser linker comprising a series of glycine and serineresidues, and optionally including additional amino acids. In otherembodiments, the linker is a lambda stump or a CH1 stump, both of whichare derived from the native sequence just after the V region in the Fab.The tascFv can be further constructed as fusion protein to contain a Fccomponent (“tascFv Fc”). In some such embodiments, such an Fc fragmentcomprises an Fc region modified to reduce or eliminate one or moreeffector functions.

The biscFv molecule is not a tandem configuration. Rather, it has a scFvat the N terminus and another at the C terminus of an Fc (“biscFv Fc”).These molecules can be made with the N terminal scFv directly fused tothe Fc hinge and with either a short or a long linker at the C terminusconnecting to the second scFv. These linkers are typically Gly-Serlinkers. In some embodiments, the Fc fragment comprises an Fc regionmodified to reduce or eliminate one or more effector functions.

The biAb molecule is also not a tandem format. It comprises a wholemonoclonal antibody with a scFv fused to the C terminus of the heavychain. These molecules can be made, for example, by converting one scFvback to a light chain (kappa or lambda) and a gamma1 heavy chain withthe second scFv connected by either a short or long Gly-Ser linker.These molecules can be made with a whole anti-IL-21 monoclonal antibodyfused to an second antigen scFv or, alternatively, with a whole secondantigen monoclonal antibody fused to an anti-IL-21 scFv. In someparticular embodiments, a biAb in accordance with the present inventioncomprises a whole anti-IL-21 monoclonal antibody (IgG1) with theC-terminal end of the heavy chain fused to an antibody specific for asecond antigen in scFv form.

Antibodies of the present invention may be described or specified interms of the epitope(s) or portion(s) of an IL-21 polypeptide of thepresent invention that they recognize or specifically bind. Theepitope(s) or polypeptide portion(s) may be specified as describedherein, e.g., by N-terminal and C-terminal positions, or by size incontiguous amino acid residues. Antibodies of the present invention mayalso be described or specified in terms of their cross-reactivity.Antibodies that do not bind any other analog, ortholog, or homolog of apolypeptide of the present invention are included.

Epitope binning refers to the use of competitive binding assays toidentify pairs of antibodies that are, or are not, capable of bindingIL-21 protein simultaneously thereby identifying antibodies that bind tothe same, or overlapping epitopes on protein. Families of antibodies (orbins) having the same or overlapping binding specificity can then beused to help define specific epitopes on IL-21. Epitope binningexperiments provide evidence that antigenically distinct epitopes arepresent. However, by themselves, they do not identify, or “map” theepitope to a specific amino acid sequence or location on the IL-21protein molecule.

Competition for binding can be evaluated for any pair of antibodies orfragments. For example, using the appropriate detection reagents, thebinding specificity of antibodies or binding fragments from anyspecies/source can be compared to the binding specificity of themonoclonal antibodies disclosed herein. Epitope binning can be performedwith “isolated antibodies” or with cell culture supernatants.Frequently, binning is performed with first round clonal supernatants toguide the choice of clones to be developed further. The antibodies to becompared should have substantially homogeneous antigen binding domains.In the case of “bispecific” or “bifunctional” antibodies the bindingspecificity of the two different binding sites need to be evaluated orbinned independently.

The present invention features ligand-specific antibodies. In additionto competitive binding of antibodies, epitope binning can also be usedto identify antibodies to either a receptor or a ligand thatcompetitively interfere with the binding of a ligand to its receptor orthe ligand mediated activation of its receptor. Frequently, favorableproperties, of a family (or bin) of antibodies can be correlated with abinding to a specific epitope defined by the epitope bin.

Competitive binding experiments do not directly measure the bindingaffinity, however the antibodies to be tested must bind sufficientlystrongly to act as competitors. Generally experimental conditions aredesigned to minimize the effects of differences in binding affinity.

Anti-IL-21 antibodies may also be useful in diagnostic assays for IL-21protein, e.g., detecting its expression in specific cells, tissues, orserum. Antibodies assigned to different bins and capable of binding todifferent immunogenic portions, or epitopes, of IL-21 may be used as thereagents for sandwich assays. In a sandwich assay, the test sampleanalyte is captured by a first antibody which is immobilized on a solidsupport, and thereafter detected by a second antibody that also binds tothe analyte, thus forming an insoluble three-part complex. See, e.g.,U.S. Pat. No. 4,376,110. The second antibody may itself be labeled witha detectable moiety (direct sandwich assays) or may be measured using ananti-immunoglobulin antibody that is labeled with a detectable moiety(indirect sandwich assay). For example, one type of sandwich assay is anELISA assay, in which case the detectable moiety is an enzyme.

The antibodies of the present invention may be assayed for specificbinding by any method known in the art. Many different competitivebinding assay formats) can be used for epitope binning. The immunoassayswhich can be used include, but are not limited to, competitive andnon-competitive assay systems using techniques such as western blots,radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich”immunoassays, immunoprecipitation assays, precipitin reactions, geldiffusion precipitin reactions, immunodiffusion assays, agglutinationassays, complement-fixation assays, immunoradiometric assays,fluorescent immunoassays, protein A immunoassays, to name just a few.Such assays are routine and well known in the art (see, e.g., Ausubel etal, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, JohnWiley & Sons, Inc., New York). Exemplary immunoassays are describedbriefly below (but are not intended by way of limitation). Additionally,a routine cross-blocking assay such as that described in Antibodies, ALaboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and DavidLane (1988), can be performed.

The BIACORE® (GE Healthcare, Piscataway, N.J.) is only one of a varietyof surface plasmon resonance assay formats that are routinely used toepitope bin panels of monoclonal antibodies. Many references (e.g. TheEpitope Mapping Protocols, Methods in Molecular Biology, Volume 66 GlennE. Morris ed. Humana Press, 1996) describe alternative methods thatcould be used to bin antibodies and would be expected to providecomparable information regarding the binding specificity of theantibodies to IL-21 protein. When using the BIACORE® system, epitopebinning experiments are performed with soluble, native or recombinantantigen. Epitope binning studies can be performed on a BIACORE1000®system (GE Healthcare, Piscataway, N.J.). BIAlogue® v. 1.2 software canbe used for programming run methods. For the example of using theBIACORE® to bin mouse monoclonal antibodies raised against IL-21,polyclonal goat anti-Mouse IgG Fc antibody (Jackson ImmunoResearchLaboratories, West Grove, Pa.) can be covalently immobilized to aBIACORE® CM5 sensor chip and used to bind (capture) the primarymonoclonal antibody of test series to the chip. Unoccupied Fc bindingsites on the chip are then blocked using a polyclonal IgG Fc fragment(Jackson ImmunoResearch Laboratories, West Grove, Pa.). Subsequently,IL-21 protein is injected and allowed to specifically bind to thecaptured primary monoclonal antibody. The BIACORE® instrument measuresthe mass of protein bound to the sensor chip, and the binding of boththe primary antibody and IL-21 antigen can be verified for each cycle.Following the binding of the primary antibody and antigen to the chip,soluble secondary antibody is injected and allowed to bind to thepre-bound antigen. If the secondary monoclonal antibody is capable ofbinding the IL-21 antigen simultaneously with the primary monoclonalantibody, its binding is detected by the BIACORE®. If, however, thesecondary monoclonal antibody is not capable of binding the IL-21antigen simultaneously with the primary monoclonal antibody, noadditional binding is detected. Each monoclonal antibody is testedagainst itself as a negative control to establish the level of thebackground (no-binding) signal.

A label-free competitive ELISA format (LFC-ELISA) can also be used tobin antibodies. This method is described by Nagata et al., J. ImmunoMethods 292:141-155, 2004. This method for epitope binning utilizedbiotinylated IL-21. For the example of binning mouse monoclonalantibodies raised against IL-21, microtiter plates are coated at 100μL/well with 1 μg/mL of a goat anti-mouse IgG Fc-γ specific antibody(Jackson ImmunoResearch) diluted in ELISA B (PBS, 0.1% Tween 20, 1%BSA). After binding of this coating antibody for 3 hours at ambienttemperature, each mAb-containing conditioned media is diluted in ELISA Bto yield an approximate mAb concentration of 0.5 μg/mL and allowed tobind to the goat anti-mouse IgG coated plates overnight at 4° C.(mAb#1). In parallel, a second set of conditioned medias (mAb#2) arediluted in polystyrene test tubes to approximately 0.5 μg/mL mAb inELISA B, mixed with 50 ng/mL biotinylated IL-21 antigen, and incubatedovernight at 4° C. After incubation of mAb#1 with the coating antibody,the plates are blocked with an unrelated antibody to saturate unoccupiedbinding sites on the plate. The mAb#2-biotin-IL-21 mixtures are added tothe plate and allowed to bind. As a control for (non-competition) in theassay, 50 ng/ml, biotinylated IL-21 is added directly (withoutpre-incubation with mAb#2) to wells containing immobilized mAb#1. Afterincubation with the biotinylated-IL-21-mAb#2 complex, streptavidin-HRP(Pierce, Rockford, Ill.) is added to the plate at 0.5 μg/mL. The platesare developed with TMB substrate (BioFX Laboratories, Owings Mills,Md.), and the absorbance of the individual wells at 450 nm is measuredwith a plate reader (Molecular Devices SPECTRAMAX®340, Sunnyvale,Calif.). If mAb#1 binds to a different epitope from mAb#2, thebiotin-IL-21-mAb#2 complex will bind to the plate resulting in a highabsorbance reading. If mAb#1 binds to the same epitope as mAb#2, thebiotin-IL-21-MAb#2 complex will not bind to the plate resulting in a lowabsorbance reading.

Ligand-specific antibodies of the present invention can simply bind toor act as antagonists of IL-21. For example, the present inventionincludes antibodies which do not disrupt IL-21's receptor/ligandinteractions or disrupt IL-21's receptor/ligand interactions eitherpartially or fully. The invention features ligand-specific antibodiesthat prevent receptor activation. The invention includes neutralizingantibodies which bind the ligand and prevent binding of the ligand tothe receptor, as well as antibodies which bind the ligand, therebypreventing receptor activation, but do not prevent the ligand frombinding the receptor. Receptor activation (i.e., signaling) may bedetermined by techniques described herein or otherwise known in the art.For example, receptor activation can be determined by detecting thephosphorylation (e.g., tyrosine or serine/threonine) of the receptor orits substrate by immunoprecipitation followed by western blot or luminexbased analysis (for example, as described supra). In specificembodiments, antibodies are provided that inhibit ligand or receptoractivity by at least 90%, at least 80%, at least 70%, at least 60%, orat least 50% of the activity in absence of the antibody.

Production of Antibodies

Antibodies to IL-21 can be generated, for example, using protein that isthe product of an IL-21 expression vector or IL-21 isolated from anatural source as an antigen. Anti-IL-21 antibodies of the presentinvention “bind specifically” to IL-21. Antibodies are considered to bespecifically binding if the antibodies exhibit at least one of thefollowing two properties: (1) antibodies bind to IL-21 with a thresholdlevel of binding activity, and (2) antibodies do not significantlycross-react with polypeptides related to IL-21. Related polypeptidescould include those of other members of the Type 1 cytokines that bindgamma common chain (γc)-containing receptors, such as IL-2, IL-4, IL-7,IL-9 and IL-15.

With regard to the first characteristic, antibodies specifically bind ifthey bind to a IL-21 polypeptide, peptide or epitope with a bindingaffinity as reflected in the measured affinity constants. To determinethe affinity characteristics, measurements of the kinetic rateconstants, equilibrium association constants, and equilibriumdissociation constants were assessed for the interaction of IL-21antagonists with the IL-21 antigen via surface plasmon resonance. Theassociation rate constant (k_(a) (M⁻¹s⁻¹)) is a value that reflects therate of the antigen-antagonist complex formation. The dissociation rateconstant (k_(d) (s⁻¹)) is a value that reflects the stability of thiscomplex. Equilibrium binding affinity is typically expressed as either adissociation equilibrium constant (K_(D) (M)) or an associationequilibrium constant (K_(A) (M⁻¹)). K_(D) is obtained by dividing thedissociation rate constant by the association rate constant(k_(d)/k_(a)), while K_(A) is obtained by dividing the association rateconstant by the dissociation rate constant (k_(a)/k_(d)). Antagonistswith similar K_(D) (or a similar K_(A)) can have widely variableassociation and dissociation rate constants. Consequently, measuring thek_(a) and k_(d) as well as the K_(A) or K_(D) helps to more uniquelydescribe the affinity of the antagonist-antigen interaction. Thepreferred affinity of an antibody is reflected in a KA (equilibriumassociation constant) of 10⁶ M⁻¹ or greater, preferably 10⁷ M⁻¹ orgreater, more preferably 10⁸ M⁻¹ or greater, and most preferably 10⁹ M⁻¹or greater. The binding affinity of an antibody can be readilydetermined by one of ordinary skill in the art, for example, byScatchard analysis (Scatchard, Ann. NY Acad. Sci. 51:660, 1949), orusing a commercially available biosensor instrument. With regard to thesecond characteristic, antibodies do not significantly cross-react withrelated polypeptide molecules, for example, if they detect IL-21, butnot other known polypeptides using a standard Western blot analysis orcapture ELISA. Examples of known related polypeptides include knownmembers of the IL-2 family to which IL-21 belongs (for example, IL-2,IL-4, IL-7, IL-9 and IL-15).

Monoclonal anti-IL-21 antibodies can be produced using antigenic IL-21epitope-bearing peptides and polypeptides. Antibodies of the presentinvention bind antigenic epitope-bearing peptides and polypeptidescontaining a sequence of at least nine, or between 15 to about 30 aminoacids contained within SEQ ID NO:2 or another amino acid sequencedisclosed herein. However, peptides or polypeptides comprising a largerportion of an amino acid sequence containing from 30 to 50 amino acids,or any length up to and including the entire amino acid sequence of apolypeptide also are useful for inducing antibodies that bind withIL-21. It is desirable that the amino acid sequence of theepitope-bearing peptide is selected to provide substantial solubility inaqueous solvents (i.e., the sequence includes relatively hydrophilicresidues, while hydrophobic residues are typically avoided). Moreover,amino acid sequences containing proline residues may be also bedesirable for large scale-antibody production.

Monoclonal anti-IL-21 antibodies can be generated by methods known tothose skilled in the alt. Rodent monoclonal antibodies to specificantigens may be obtained by known methods (see, for example, Kohler etal., Nature 256:495 (1975), Coligan et al. (eds.), Current Protocols inImmunology, Vol. 1, pages 2.5.1-2.6.7 (John Wiley & Sons 1991)[“Coligan”], Picksley et al., “Production of monoclonal antibodiesagainst proteins expressed in E. coli,” in DNA Cloning 2: ExpressionSystems, 2nd Edition, Glover et al. (eds.), page 93 (Oxford UniversityPress 1995)).

Antibodies to a second antigen, related to autoimmune disease, can begenerated, for example, using protein that is the product of anexpression vector or that antigen isolated from a natural source.Antibodies to the second antigen that comprise the present invention“bind specifically” to their antigen. Antibodies are considered to bespecifically binding if the antibodies exhibit at least one of thefollowing two properties: (1) antibodies bind to the antigen with athreshold level of binding activity, and (2) antibodies do notsignificantly cross-react with polypeptides related to the antigen.Related polypeptides could include those of other members of theantigen's protein family.

With regard to the first characteristic, antibodies specifically bind ifthey bind to a the second antigen polypeptide, peptide or epitope with abinding affinity as reflected in the measured affinity constants. Todetermine the affinity characteristics, measurements of the kinetic rateconstants, equilibrium association constants, and equilibriumdissociation constants were assessed for the interaction of secondantigen antagonists with the second antigen via surface plasmonresonance. The association rate constant (k_(a) (M⁻¹s⁻¹)) is a valuethat reflects the rate of the antigen-antagonist complex formation. Thedissociation rate constant (k_(d) (s⁻¹)) is a value that reflects thestability of this complex. Equilibrium binding affinity is typicallyexpressed as either a dissociation equilibrium constant (K_(D) (M)) oran association equilibrium constant (K_(A) (M⁻¹)). K_(D) is obtained bydividing the dissociation rate constant by the association rate constant(k_(d)/k_(a)), while K_(A) is obtained by dividing the association rateconstant by the dissociation rate constant (k_(a)/k_(d)). Antagonistswith similar K_(D) (or a similar K_(A)) can have widely variableassociation and dissociation rate constants. Consequently, measuring thek_(a) and k_(d) as well as the K_(A) or K_(D) helps to more uniquelydescribe the affinity of the antagonist-antigen interaction. Thepreferred affinity of an antibody is reflected in a KA (equilibriumassociation constant) of 10⁶ M⁻¹ or greater, preferably 10⁷ M⁻¹ orgreater, more preferably 10⁸ M⁻¹ or greater, and most preferably 10⁹ M⁻¹or greater. The binding affinity of an antibody can be readilydetermined by one of ordinary skill in the art, for example, byScatchard analysis (Scatchard, Ann. NY Acad. Sci. 51:660, 1949), orusing a commercially available biosensor instrument. With regard to thesecond characteristic, antibodies do not significantly cross-react withrelated polypeptide molecules, for example, if they detect the secondantigen, but not other known polypeptides using a standard Western blotanalysis or capture ELISA.

Monoclonal anti-second antigen antibodies can be produced usingantigenic epitope-bearing peptides and polypeptides of that antigen.Antibodies of the present invention bind antigenic epitope-bearingpeptides and polypeptides containing a sequence of at least nine, orbetween 15 to about 30 amino acids of its protein sequence. However,peptides or polypeptides comprising a larger portion of an amino acidsequence containing from 30 to 50 amino acids, or any length up to andincluding the entire amino acid sequence of a polypeptide also areuseful for inducing antibodies that bind with the second antigen. It isdesirable that the amino acid sequence of the epitope-bearing peptide isselected to provide substantial solubility in aqueous solvents (i.e.,the sequence includes relatively hydrophilic residues, while hydrophobicresidues are typically avoided). Moreover, amino acid sequencescontaining proline residues may be also be desirable for largescale-antibody production.

Monoclonal anti-second antigen antibodies can be generated by methodsknown to those skilled in the art. Rodent monoclonal antibodies tospecific antigens may be obtained by known methods (see, for example,Kohler et al., Nature 256:495 (1975), Coligan et al. (eds.), CurrentProtocols in Immunology, Vol. 1, pages 2.5.1-2.6.7 (John Wiley & Sons1991) [“Coligan”], Picksley et al., “Production of monoclonal antibodiesagainst proteins expressed in E. coli,” in DNA Cloning 2: ExpressionSystems, 2nd Edition, Glover et al. (eds.), page 93 (Oxford UniversityPress 1995)).

The antibodies of the invention for the second antigen can be producedby any method known in the art for the synthesis of antibodies, inparticular, by chemical synthesis or preferably, by recombinantexpression techniques. Recombinant expression of an antibody of theinvention, or fragment, derivative or analog thereof, e.g., a heavy orlight chain of an antibody of the invention, requires construction of anexpression vector containing a polynucleotide that encodes the antibody.Once a polynucleotide encoding an antibody molecule or a heavy or lightchain of an antibody, or portion thereof (preferably containing theheavy or light chain variable domain), of the invention has beenobtained, the vector for the production of the antibody molecule may beproduced by recombinant DNA technology using techniques well known inthe art. Thus, methods for preparing a protein by expressing apolynucleotide containing an antibody encoding nucleotide sequence aredescribed herein. Methods which are well known to those skilled in theart can be used to construct expression vectors containing antibodycoding sequences and appropriate transcriptional and translationalcontrol signals. These methods include, for example, in vitrorecombinant DNA techniques, synthetic techniques, and in vivo geneticrecombination. The invention, thus, provides replicable vectorscomprising a nucleotide sequence encoding an antibody molecule of theinvention, or a heavy or light chain thereof, or a heavy or light chainvariable domain, operably linked to a promoter. Such vectors may includethe nucleotide sequence encoding the constant region of the antibodymolecule (see, e.g., PCT Publication WO 86/05807; PCT Publication WO89/01036; and U.S. Pat. No. 5,122,464) and the variable domain of theantibody may be cloned into such a vector for expression of the entireheavy or light chain.

The expression vector is transferred to a host cell by conventionaltechniques and the transfected cells are then cultured by conventionaltechniques to produce an antibody to a second antigen of the invention.Thus, the invention includes host cells containing a polynucleotideencoding an antibody of the invention, or a heavy or light chainthereof, operably linked to a heterologous promoter. In preferredembodiments for the expression of double-chained antibodies, vectorsencoding both the heavy and light chains may be co-expressed in the hostcell for expression of the entire immunoglobulin molecule, as detailedbelow.

A variety of host-expression vector systems may be utilized to expressthe antibody molecules of the invention, including those thatspecifically bind the second antigen. Such host-expression systemsrepresent vehicles by which the coding sequences of interest may beproduced and subsequently purified, but also represent cells which may,when transformed or transfected with the appropriate nucleotide codingsequences, express an antibody molecule of the invention in situ. Theseinclude but are not limited to microorganisms such as bacteria (e.g., E.coli, B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containing antibody codingsequences; yeast (e.g., Saccharomyces, Pichia) transformed withrecombinant yeast expression vectors, containing antibody codingsequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing antibody codingsequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing antibody coding sequences; or mammalian cellsystems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinantexpression constructs containing promoters derived from the genome ofmammalian cells (e.g., metallothionein promoter) or from mammalianviruses (e.g., MPSV, CMV, the adenovirus late promoter; the vacciniavirus 7.5K promoter). Preferably, bacterial cells such as Escherichiacoli, and more preferably, eukaryotic cells, especially for theexpression of whole recombinant antibody molecule, are used for theexpression of a recombinant antibody molecule. For example, mammaliancells such as Chinese hamster ovary cells (CHO), in conjunction with avector such as the major intermediate early gene promoter element fromhuman cytomegalovirus, CMV enhancer or MPSV promoter is an effectiveexpression system for antibodies (Foecking et al., 1986, Gene 45:101;Cockett et al., 1990, Bio/Technology 8:2).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified may be desirable. Such vectors include, but are not limited, tothe E. coli expression vector pUR278 (Ruther et al., 1983, EMBO J.2:1791), in which the antibody coding sequence may be ligatedindividually into the vector in frame with the lacZ coding region sothat a fusion protein is produced; pIN vectors (Inouye & Inouye, NucleicAcids Res. 13:3101-3109, 1985; Van Heeke & Schuster, J. Biol. Chem.24:5503-5509, 1989); and the like. pGEX vectors may also be used toexpress foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption and binding to amatrix glutathione-agarose beads followed by elution in the presence offree glutathione. The pGEX vectors are designed to include thrombin orfactor Xa protease cleavage sites so that the cloned target gene productcan be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The antibody coding sequence may be clonedindividually into non-essential regions (for example the polyhedringene) of the virus and placed under control of an AcNPV promoter (forexample the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the antibody coding sequence of interest may be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene may then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing the antibody molecule in infected hosts. (e.g., see Logan &Shenk, Proc. Natl. Acad. Sci. USA 81:355-359, 1984). Specific initiationsignals may also be required for efficient translation of insertedantibody coding sequences. These signals include the ATG initiationcodon and adjacent sequences. Furthermore, the initiation codon must bein phase with the reading frame of the desired coding sequence to ensuretranslation of the entire insert. These exogenous translational controlsignals and initiation codons can be of a variety of origins, bothnatural and synthetic. The efficiency of expression may be enhanced bythe inclusion of appropriate transcription enhancer elements,transcription terminators, etc. (see Bittner et al., Methods in Enzymol.153:51-544, 1987).

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used. Such mammalian hostcells include but are not limited to CHO, VERO, BHK, Hela, COS, MDCK,293, 3T3, WI38, and in particular, breast cancer cell lines such as, forexample, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary glandcell line such as, for example, CRL7030 and Hs578Bst.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe antibody molecule may be engineered. Rather than using expressionvectors which contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express the antibodymolecule. Such engineered cell lines may be particularly useful inscreening and evaluation of compounds that interact directly orindirectly with the antibody molecule.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223,1977), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, Proc. Natl. Acad. Sci. USA 48:202, 1992), and adeninephosphoribosyltransferase (Lowy et al., Cell 22:817, 1980) genes can beemployed in tk-, hgprt- or aprt-cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., Proc. Natl. Acad. Sci. USA 77:357, 1980; O'Hare et al., Proc.Natl. Acad. Sci. USA 78:1527, 1981); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072,1981); neo, which confers resistance to the aminoglycoside G-418 (Wu andWu, Biotherapy 3:87-95, 1991; Tolstoshev, Ann. Rev. Pharmacol. Toxicol.32:573-596, 1993; Mulligan, Science 260:926-932, 1993; and Morgan andAnderson, Ann. Rev. Biochem. 62:191-217, 1993; TIB TECH 11(5):155-215),May, 1993; and hygro, which confers resistance to hygromycin (Santerreet al., Gene 30:147, 1984). Methods commonly known in the art ofrecombinant DNA technology which can be used are described in Ausubel etal. (eds.), 1993, Current Protocols in Molecular Biology, John Wiley &Sons, NY; Kriegler, 1990, Gene Transfer and Expression, A LaboratoryManual, Stockton Press, NY; and in Chapters 12 and 13, Dracopoli et al.(eds), 1994, Current Protocols in Human Genetics, John Wiley & Sons,NY.; Colberre-Garapin et al., J. Mol. Biol. 150:1, 1981; which areincorporated by reference herein in their entireties.

The expression levels of an antibody molecule can be increased by vectoramplification (for a review, see Bebbington and Hentschel, The use ofvectors based on gene amplification for the expression of cloned genesin mammalian cells in DNA cloning, Vol. 3. (Academic Press, New York,1987)). When a marker in the vector system expressing antibody isamplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the antibody gene, production ofthe antibody will also increase (Crouse et al., Mol. Cell. Biol. 3:257,1983).

The host cell may be co-transfected with two expression vectors of theinvention, the first vector encoding a heavy chain derived polypeptideand the second vector encoding a light chain derived polypeptide. Thetwo vectors may contain identical selectable markers which enable equalexpression of heavy and light chain polypeptides. Alternatively, asingle vector may be used which encodes both heavy and light chainpolypeptides. In such situations, the light chain should be placedbefore the heavy chain to avoid an excess of toxic free heavy chain(Proudfoot, Nature 322:52, 1986; Kohler, Proc. Natl. Acad. Sci. USA77:2197, 1980). The coding sequences for the heavy and light chains maycomprise cDNA or genomic DNA.

Once an antibody molecule of the invention has been recombinantlyexpressed, it may be purified by any method known in the art forpurification of an immunoglobulin molecule, for example, bychromatography (e.g., ion exchange, affinity, particularly by affinityfor the specific antigen after Protein A, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins.

For particular uses, it may be desirable to prepare fragments ofanti-IL-21 antibodies. Such antibody fragments can be obtained, forexample, by proteolytic hydrolysis of the antibody. Antibody fragmentscan be obtained by pepsin or papain digestion of whole antibodies byconventional methods. As an illustration, antibody fragments can beproduced by enzymatic cleavage of antibodies with pepsin to provide a 5Sfragment denoted F(ab′)2. This fragment can be further cleaved using athiol reducing agent to produce 3.5S Fab′ monovalent fragments.Optionally, the cleavage reaction can be performed using a blockinggroup for the sulfhydryl groups that result from cleavage of disulfidelinkages. As an alternative, an enzymatic cleavage using pepsin producestwo monovalent Fab fragments and an Fc fragment directly. These methodsare described, for example, by Goldenberg, U.S. Pat. No. 4,331,647,Nisonoff et al., Arch Biochem. Biophys. 89:230, 1960; Porter, Biochem.J. 73:119, 1959; Edelman et al., in Methods in Enzymology Vol. 1, page422 (Academic Press 1967), and by Coligan at pages 2.8.1-2.8.10 and2.10.-2.10.4.

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

For example, Fv fragments comprise an association of VH and VL chains.This association can be noncovalent, as described by Inbar et al., Proc.Nat'l Acad. Sci. USA 69:2659, 1972. Alternatively, the variable chainscan be linked by an intermolecular disulfide bond or cross-linked bychemicals such as glutaraldehyde (see, for example, Sandhu, Crit. Rev.Biotech. 12:437, 1992).

The Fv fragments may comprise VH and VL chains which are connected by apeptide linker. These single-chain antigen binding proteins (scFv) areprepared by constructing a structural gene comprising DNA sequencesencoding the VH and VL domains which are connected by anoligonucleotide. The structural gene is inserted into an expressionvector which is subsequently introduced into a host cell, such as E.coli. The recombinant host cells synthesize a single polypeptide chainwith a linker peptide bridging the two V domains. Methods for producingscFvs are described, for example, by Whitlow et al.; Methods: ACompanion to Methods in Enzymology 2:97 (1991) (also see, Bird et al.,Science 242:423, 1988, Ladner et al., U.S. Pat. No. 4,946,778, Pack etal., Bio/Technology 11:1271, 1993, and Sandhu, supra).

It is also possible to construct alternative frameworks by using acollection of monomeric proteins to form a monomer domain. These monomerdomains can be small enough to penetrate tissues. The monomer domainscan be naturally-occurring or non-natural variants or combinationthereof. Monomer domains can form multimers of two or more domains. Themonomer domain binds a position, analogous to epitopes described herein,on a target molecule. In some cases, the multimer can be formed from avariety of monomer domains. (See, e.g. U.S. Patent Application2004-0132028 and U.S. Patent Application 2006-0177831.)

The antibodies of the present invention include derivatives that aremodified, i.e, by the covalent attachment of any type of molecule to theantibody such that covalent attachment does not prevent the antibodyfrom binding IL-21 or blocking receptor activation or from binding thesecond antigen, if the antibody is bispecific. For example, but not byway of limitation, the antibody derivatives include antibodies that havebeen modified, e.g., by glycosylation, acetylation, pegylation,phosphylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. Any of numerous chemical modifications may be carried outby known techniques, including, but not limited to specific chemicalcleavage, acetylation, formylation, metabolic synthesis of tunicamycin,etc. Additionally, the derivative may contain one or more non-classicalamino acids.

An anti-IL-21 antibody or second antigen antibody can be conjugated witha detectable label to form an anti-IL-21 immunoconjugate. Suitabledetectable labels include, for example, a radioisotope, a fluorescentlabel, a chemiluminescent label, an enzyme label, a bioluminescent labelor colloidal gold. Methods of making and detecting suchdetectably-labeled immunoconjugates are well-known to those of ordinaryskill in the art, and are described in more detail below. The detectablelabel can be a radioisotope that is detected by autoradiography.Isotopes that are particularly useful for the purpose of the presentinvention are ³H, ¹²⁵I, ¹³¹I, ³⁵S and ¹⁴C.

Anti-IL-21 or second antigen antibody immunoconjugates can also belabeled with a fluorescent compound. The presence of afluorescently-labeled antibody is determined by exposing theimmunoconjugate to light of the proper wavelength and detecting theresultant fluorescence. Fluorescent labeling compounds includefluorescein isothiocyanate, rhodamine, phycoerytherin, phycocyanin,allophycocyanin, o-phthaldehyde, alexadyes, fluorescent nonparticles(e.g. Q dots) and fluorescamine.

It is also possible that anti-IL-21 immunoconjugates or second antigenimmunoconjugates can be detectably labeled by coupling an antibodycomponent to a chemiluminescent compound. The presence of thechemiluminescent-tagged immunoconjugate is determined by detecting thepresence of luminescence that arises during the course of a chemicalreaction. Examples of chemiluminescent labeling compounds includeluminol, isoluminol, an aromatic acridinium ester, an imidazole, anacridinium salt and an oxalate ester.

Similarly, a bioluminescent compound can be used to label anti-IL-21 orsecond antibody immunoconjugates of the present invention.Bioluminescence is a type of chemiluminescence found in biologicalsystems in which a catalytic protein increases the efficiency of thechemiluminescent reaction. The presence of a bioluminescent protein isdetermined by detecting the presence of luminescence. Bioluminescentcompounds that are useful for labeling include luciferin, luciferase andaequorin.

Alternatively, anti-IL-21 or second antibody immunoconjugates can bedetectably labeled by linking an anti-IL-21 antibody component to anenzyme. When the anti-IL-21-enzyme conjugate is incubated in thepresence of the appropriate substrate, the enzyme moiety reacts with thesubstrate to produce a chemical moiety which can be detected, forexample, by spectrophotometric, fluorometric or visual means. Examplesof enzymes that can be used to detectably label polyspecificimmunoconjugates include β-galactosidase, glucose oxidase, peroxidaseand alkaline phosphatase.

Those of skill in the art will know of other suitable labels which canbe employed in accordance with the present invention. The binding ofmarker moieties to anti-IL-21 antibodies can be accomplished usingstandard techniques known to the art. Typical methodology in this regardis described by the following: Kennedy et al., Clin. Chim. Acta 70:1,1976; Schurs et al., Clin. Chim. Acta 81:1, 1977; Shih et al., Int'l J.Cancer 46:1101, 1990; Stein et al., Cancer Res. 50:1330, 1990; andColigan, supra.

Moreover, the convenience and versatility of immunochemical detectioncan be enhanced by using anti-IL-21 or second antibody antibodies thathave been conjugated with avidin, streptavidin, and biotin (see, forexample, Wilchek et al. (eds.), “Avidin-Biotin Technology,” Methods InEnzymology, Vol. 184 (Academic Press 1990), and Bayer et al.,“Immunochemical Applications of Avidin-Biotin Technology,” in Methods InMolecular Biology, Vol. 10, Manson (ed.), pages 149-162 (The HumanaPress, Inc. 1992).

Methods for performing immunoassays are well-established. See, forexample, Cook and Self, “Monoclonal Antibodies in DiagnosticImmunoassays,” in Monoclonal Antibodies: Production, Engineering, andClinical Application, Ritter and Ladyman (eds.), pages 180-208,(Cambridge University Press, 1995), Perry, “The Role of MonoclonalAntibodies in the Advancement of Immunoassay Technology,” in MonoclonalAntibodies: Principles and Applications, Birch and Lennox (eds.), pages107-120 (Wiley-Liss, Inc. 1995), and Diamandis, Immunoassay (AcademicPress, Inc. 1996).

Antibodies or fragments thereof having increased in vivo half-lives canbe generated by techniques known to those of skill in the art. Forexample, antibodies or fragments thereof with increased in vivohalf-lives can be generated by modifying (e.g., substituting, deletingor adding) amino acid residues identified as involved in the interactionbetween the Fc domain and the FcRn receptor (see, e.g., InternationalPublication Nos. WO 97/34631 and WO 02/060919, which are incorporatedherein by reference in their entireties). Antibodies or fragmentsthereof with increased in vivo half-lives can be generated by attachingto said antibodies or antibody fragments polymer molecules such as highmolecular weight polyethyleneglycol (PEG). PEG can be attached to saidantibodies or antibody fragments with or without a multifunctionallinker either through site-specific conjugation of the PEG, for example,to the N- or C-terminus of said antibodies or antibody fragments or viaepsilon-amino groups present on lysine residues. Linear or branchedpolymer derivatization that results in minimal loss of biologicalactivity will be used. The degree of conjugation will be closelymonitored by SDS-PAGE and mass spectrometry to ensure proper conjugationof PEG molecules to the antibodies. Unreacted PEG can be separated fromantibody-PEG conjugates by, e.g., size exclusion or ion-exchangechromatography.

Pharmaceutical Compositions

The present invention further includes pharmaceutical compositions,comprising a pharmaceutically acceptable carrier and one or morepolypeptide or antibody described herein. The pharmaceutical compositioncan include additional therapeutic agents, including but not limited tocytotoxic agents a cytotoxin, e.g., a cytostatic or cytocidal agent, atherapeutic agent or a radioactive metal ion. A cytotoxin or cytotoxicagent includes any agent that is detrimental to cells. Examples includepaclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin,doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, and puromycin and analogsor homologs thereof. Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxombicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine). For example, the pharmaceuticalcomposition can comprise a protein or polypeptide possessing a desiredbiological activity. Such proteins may include, for example, a toxinsuch as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; aprotein such as tumor necrosis factor, α-IFN, β-IFN, nerve growthfactor, platelet derived growth factor, tissue plasminogen activator, athrombotic agent or an anti-angiogenic agent, e.g., angiostatin orendostatin; or, biological response modifiers such as, for example,lymphokines, interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6(IL-6), granulocyte macrophage colony stimulating factor (GM-CSF),granulocyte colony stimulating factor (G-CSF), antibodies designed toantagonize biological response modifiers, other antibodies, other Fcfusion proteins or other growth factors.

For purposes of therapy, anti-IL-21 antibody molecules and apharmaceutically acceptable carrier are administered to a patient in atherapeutically effective amount. This administered composition can alsocomprise a second antibody to autoimmune related antigen, as describedabove. A combination of a therapeutic molecule of the present inventionand a pharmaceutically acceptable carrier is said to be administered ina “therapeutically effective amount” if the amount administered isphysiologically significant. An agent is physiologically significant ifits presence results in a detectable change in the physiology of arecipient patient. For example, an agent used to treat inflammation isphysiologically significant if its presence alleviates the inflammatoryresponse.

Degradable polymer microspheres have been designed to maintain highsystemic levels of therapeutic proteins. Microspheres are prepared fromdegradable polymers such as poly(lactide-co-glycolide) (PLG),polyanhydrides, poly(ortho esters), nonbiodegradable ethylvinyl acetatepolymers, in which proteins are entrapped in the polymer (Gombotz andPettit, Bioconjugate Chem. 6:332, 1995; Ranade, “Role of Polymers inDrug Delivery,” in Drug Delivery Systems, Ranade and Hollinger (eds.),pages 51-93 (CRC Press 1995); Roskos and Maskiewicz, “DegradableControlled Release Systems Useful for Protein Delivery,” in ProteinDelivery: Physical Systems, Sanders and Hendren (eds.), pages 45-92(Plenum Press 1997); Bartus et al., Science 281:1161, 1998; Putney andBurke, Nature Biotechnology 16:153, 1998; Putney, Curr. Opin. Chem.Biol. 2:548, 1998). Polyethylene glycol (PEG)-coated nanospheres canalso provide carriers for intravenous administration of therapeuticproteins (see, for example, Gref et al., Pharm. Biotechnol. 10:167,1997).

Other dosage forms can be devised by those skilled in the art, as shown,for example, by Ansel and Popovich, Pharmaceutical Dosage Forms and DrugDelivery Systems, 5th Edition (Lea & Febiger 1990), Gennaro (ed.),Remington's Pharmaceutical Sciences, 19th Edition (Mack PublishingCompany 1995), and by Ranade and Hollinger, Drug Delivery Systems (CRCPress 1996).

Pharmaceutical compositions may be supplied as a kit comprising acontainer that comprises a neutralizing anti-IL-21 antibody. The kit canalso comprise an antibody to a second autoimmune disease related antigenas described above. Therapeutic polypeptides can be provided in the formof an injectable solution for single or multiple doses, or as a sterilepowder that will be reconstituted before injection. Alternatively, sucha kit can include a dry-powder disperser, liquid aerosol generator, ornebulizer for administration of a therapeutic polypeptide. Such a kitmay further comprise written information on indications and usage of thepharmaceutical composition.

A pharmaceutical composition comprising anti-IL-21 antibodies and/or anadditional second antibody can be furnished in liquid form, in anaerosol, or in solid form. Liquid forms, are illustrated by injectablesolutions, aerosols, droplets, topological solutions and oralsuspensions. Exemplary solid forms include capsules, tablets, andcontrolled-release forms. The latter form is illustrated by miniosmoticpumps and implants (Bremer et al., Pharm. Biotechnol. 10:239, 1997;Ranade, “Implants in Drug Delivery,” in Drug Delivery Systems, Ranadeand Hollinger (eds.), pages 95-123 (CRC Press 1995); Bremer et al.,“Protein Delivery with Infusion Pumps,” in Protein Delivery: PhysicalSystems, Sanders and Hendren (eds.), pages 239-254 (Plenum Press 1997);Yewey et al., “Delivery of Proteins from a Controlled Release InjectableImplant,” in Protein Delivery: Physical Systems, Sanders and Hendren(eds.), pages 93-117 (Plenum Press 1997)). Other solid forms includecreams, pastes, other topological applications, and the like.

Therapeutic Uses for Anti-IL-21 Antibodies or Anti-IL-21 and SecondAntigen Bispecific Antibodies

IL-21 is a CD4⁺ T cell-derived cytokine that is important for optimalCD8⁺ T cell mediated immunity, NK cell activation, and optimal humoralresponses, such as antibody production and B cell maturation. IL-21 hasbeen shown to induce a number of proinflammatory chemokines andcytokines, such as IL-18, IL-15, IL-5, IL-6, IL-7A, IL-17F, TNFRII,sCD25, and RANTES. IL-21 also induces an acute phase response innon-human primates and humans when administered by IV or SC injection(Dodds et al., Cancer Immunol Immunother 2008 Oct. 17 [electronicpublication]). In vitro, stimulates the growth of certain neoplasticimmune cell populations such as multiple myeloma cells and acute T-cellleukemia (Brenne et al Blood 99(10):3756-62 (2002), diCarlo E, et alCancer Immunol Immunother 56(9):1323-1324 (2007)). IL-21 is alsoproduced by Hodgkin Reed-Sternberg cells in Hodgkin's Lymphoma(Lamprecht et al., Blood 112(8):3339-47, 2008). Increased expression ofIL-21 receptor has been shown in epidermis in patients with systemicsclerosis (Distler et al., Arthritis & Rheumatism 52:865-864, 2004) andrheumatoid arthritis synovial fibroblasts (Jungel et al., Arthritis &Rheumatism 50:1468-1476, 2004). Moreover, autoimmune, diabetic NOD micehave increased EL-21 receptor expression (King et al., Cell 117:265-277,2004.) It has been shown that IgG and IL-21 expression is increased inthe BXSB-Yaa mouse model which develop an autoimmune lupuserythematosus-like disease (Ozaki et al., J. Immunol. 173:5361-5371,2004); IL-21 expression is higher in lupus-prone sanroque mice (Vinuesaet al. Nature 435:452, 2005); IL-21 expression is higher in inflamed vsuninflamed gut tissues from patients with Crohn's disease (Monteleone,et al., Gastroenterology 128:687-694, 2005). IL-21 is also overproducedin the mucosa of celiac disease patients (Finn et al. Gut,PMID:17965065, 2007).

A therapeutically effective amount of an anti-IL-21 antibody and/or anantibody to a second antigen refers to an amount of antibody which whenadministered to a subject is effective to prevent, delay, reduce orinhibit a symptom or biological activity associated with a disease ordisorder. Administration may consist of a single dose or multiple dosesand may be given in combination with other pharmaceutical compositions.

The present invention provides compositions and methods for usinganti-IL-21 monoclonal antibodies as well as bispecific antibodiescomprising those antibodies in inflammatory and immune diseases orconditions such as psoriasis, pancreatitis, type I diabetes (IDDM),Graves' Disease, inflammatory bowel disease (IBD), Crohn's Disease,ulcerative colitis, irritable bowel syndrome, multiple sclerosis,rheumatoid arthritis, reactive arthritis, enteropathic arthritis,spondyloarthropathy, autoimmune myocarditis, Kawasaki disease, celiacdisease, uveitis, Behcet's disease, coronary artery disease, chronicobstructive pulmonary disease (COPD), interstitial lung disease,inflammatory muscle disease (polymyositis, dermatomyositis), microscopicpolyangiitis, autoimmune aplastic anemia, autoimmune thyroiditis,autoimmune hepatitis, Wegener's syndrome, diverticulosis, systemic lupuserythematosus, ankylosing spondylitis, scleroderma, systemic sclerosis,psoriatic arthritis, osteoarthritis, atopic dermatitis, vitiligo, graftvs. host disease (GVHD), cutaneous T cell lymphoma (CTCL), Sjogren'ssyndrome, glomerulonephritis, IgA nephropathy, autoimmune nephritis,pemphigus vulgaris, myasthenia gravis, autoimmune hearing loss,neuromyelitis optica, Goodpasture's syndrome, cryoglobulinemia, GuillainBarre syndrome, chronic inflammatory demyelinating polyneuropathy(CIDP), autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura(ITP), transplant rejection, highly sensitized transplant patients,anti-phospholipid syndrome, allergy, and asthma, and other autoimmunediseases. The present invention provides compositions and methods forusing anti-IL-21 monoclonal antibodies in the therapy for certain immunecell cancers such as multiple myeloma, acute T-cell leukemia, orHodgkin's Lymphoma.

Contact Dermatitis

Allergic contact dermatitis is defined as a T cell mediated immunereaction to an antigen that comes into contact with the skin. The CLA+ Tcell population is believed to be involved in the initiation ofdermatitis since allergen dependent T cell responses are largelyconfined to the CLA+ population of cells (See Santamaria-Babi, et al., JExp Med 181:1935, (1995)). Recent data have found that only memory(CD45RO+) CD4+ CLA+ and not CD8+ T cells proliferate and produce bothtype-1 (IFN-γ) and type-2 (IL-5) cytokines in response to nickel, acommon contact hypersensitivity allergen. Furthermore, cells expressingCLA in combination with CD4, CD45RO (memory) or CD69 are increased afternickel-specific stimulation and express the chemokine receptors CXCR3,CCR4, CCR10 but not CCR6. See Moed et al., Br J Dermatol 51:32, (2004).

In animal models, it has been demonstrated that allergic contactdermatitis is T cell-dependent and that the allergic-responsive T cellsmigrate to the site of allergen application. See generally: Engeman, etal., J Immunol 164:5207, (2000); Ferguson & Kupper, J Immunol 150:1172,(1993); and Gorbachev & Fairchild, Crit Rev Immunol. 21:451 (2001).

Administration anti-IL-21 antibodies to mousse models of contacthypersensitivity is used to evaluate the clinical utility of anti-Th-21antibodies to ameliorate symptoms and alter the course of disease. Theaddition of a second antibody to an antigen known to be involved incontact dermatitis can be used. A bispecific antibody is particularlypreferred if addition of the second antigen increases efficacy of thetreatment and/or increases specificity of the antibody binding.

Atopic Dermatitis

Atopic dermatitis (AD) is a chronically relapsing inflammatory skindisease with a dramatically increasing incidence over the last decades.Clinically AD is characterized by highly pruritic, often excoriated,plaques and papules that show a chronic relapsing course. The diagnosisof AD is mostly based on major and minor clinical findings. See HardinJ. M., Arch Dermatol 135:1551 (1999). Histopathology reveals spongiosis,hyperparakeratosis and focal parakeratosis in acute lesions, whereasmarked epidermal hyperplasia with hyperparakeratosis and parakeratosis,acanthosis/hypergranulosis and perivascular infiltration of the dermiswith lymphocytes and abundant mast cells are the hallmarks of chroniclesions.

T cells play a central role in the initiation of local immune responsesin tissues and evidence suggests that skin-infiltrating T cells inparticular, may play a key role in the initiation and maintenance ofdisregulated immune responses in the skin. Approximately 90% ofinfiltrating T cells in cutaneous inflammatory sites express thecutaneous lymphocyte-associated antigen which binds E-selectin, aninducible adhesion molecule on endothelium (reviewed in Santamaria-Babi,et al., Eur J Dermatol 14:13, (2004)). A significant increase incirculating CLA+ T cells among AD patients compared with controlindividuals has been documented (See Teraki, et al., Br J Dermatol143:373 (2000), while others have demonstrated that memory CLA+ T cellsfrom AD patients preferentially respond to allergen extract compared tothe CLA− population (See Santamaria-Babi, L. F., et al., J Exp Med.181:1935, (1995)). In humans, the pathogenesis of atopic disorders ofthe skin have been associated with increases in CLA+ T cells thatexpress increased levels of Th-2-type cytokines like IL-5 and IL-13. SeeAkdis et al., Eur J Immunol 30:3533 (2000); and Hamid et al., J AllergyClin Immunol 98: 225 (1996).

NC/Nga mice spontaneously develop AD-like lesions that parallel human ADin many aspects, including clinical course and signs, histopathology andimmunopathology when housed in non-specified pathogen-free (non-SPF)conditions at around 6-8 weeks of age. In contrast, NC/Nga mice keptunder SPF conditions do not develop skin lesions. However, onset ofspontaneous skin lesions and scratching behaviour can be synchronized inNC/Nga mice housed in a SPF facility by weekly intradermal injection ofcrude dust mite antigen. See Matsuoka et al., Allergy 58:139 (2003).Therefore, the development of AD in NC/Nga is a useful model for theevaluation of novel therapeutics for the treatment of AD.

In addition to the NC/Nga model of spontaneous AD, epicutaneoussensitization of mice using OVA can also be used as a model to induceantigen-dependent epidermal and dermal thickening with a mononuclearinfiltrate in skin of sensitized mice. This usually coincides withelevated serum levels of total and specific IgE, however no skin barrierdysfunction or pruritus normally occurs in this model. See Spergel etal., J Clin Invest, 101:1614, (1998). This protocol can be modified inorder to induce skin barrier disregulation and pruritis by sensitizingDO11.10 OVA TCR transgenic mice with OVA. Increasing the number ofantigen-specific T cells that could recognize the sensitizing antigenmay increase the level of inflammation in the skin to induce visiblescratching behaviour and lichenification/scaling of the skin.

Administration of anti-IL-21 antibodies to mouse models of atopicdermatitis is used to evaluate the clinical utility of anti-IL-21antibodies to ameliorate symptoms and alter the course of disease. Theaddition of a second antibody to an antigen known to be involved inatopic dermatitis can be used. A bispecific antibody is particularlypreferred if addition of the second antigen increases efficacy of thetreatment and/or increases specificity of the antibody binding

Arthritis

Arthritis, including osteoarthritis, rheumatoid arthritis, arthriticjoints as a result of injury, and the like, are common inflammatoryconditions which would benefit from the therapeutic use ofanti-inflammatory antibodies and binding polypeptides. For example,rheumatoid arthritis (RA) is a systemic disease that affects the entirebody and is one of the most common forms of arthritis. It ischaracterized by the inflammation of the membrane lining the joint,which causes pain, stiffness, warmth, redness and swelling. Inflammatorycells release enzymes that may digest bone and cartilage. As a result ofrheumatoid arthritis, the inflamed joint lining, the synovium, caninvade and damage bone and cartilage leading to joint deterioration andsevere pain amongst other physiologic effects. The involved joint canlose its shape and alignment, resulting in pain and loss of movement.

Rheumatoid Arthritis

Rheumatoid arthritis (RA) is an immune-mediated disease particularlycharacterized by inflammation and subsequent tissue damage leading tosevere disability and increased mortality. A variety of cytokines areproduced locally in the rheumatoid joints. Numerous studies havedemonstrated that IL-1 and TNF-alpha, two prototypic pro-inflammatorycytokines, play an important role in the mechanisms involved in synovialinflammation and in progressive joint destruction. Indeed, theadministration of TNF-alpha and IL-1 inhibitors in patients with RA hasled to a dramatic improvement of clinical and biological signs ofinflammation and a reduction of radiological signs of bone erosion andcartilage destruction. However, despite these encouraging results, asignificant percentage of patients do not respond to these agents,suggesting that other mediators are also involved in the pathophysiologyof arthritis (Gabay, Expert. Opin. Biol. Ther. 2(2):135-149, 2002).

There are several animal models for rheumatoid arthritis known in theart. For example, in the collagen-induced arthritis (CIA) model, micedevelop chronic inflammatory arthritis that closely resembles humanrheumatoid arthritis. Since CIA shares similar immunological andpathological features with RA, this makes it an ideal model forscreening potential human anti-inflammatory compounds. The CIA model isa well-known model in mice that depends on both an immune response, andan inflammatory response, in order to occur. The immune responsecomprises the interaction of B-cells and CD4+ T-cells in response tocollagen, which is administered as antigen, and leads to the productionof anti-collagen antibodies. The inflammatory phase is the result oftissue responses from mediators of inflammation, as a consequence ofsome of these antibodies cross-reacting to the mouse's native collagenand activating the complement cascade. An advantage in using the CIAmodel is that the basic mechanisms of pathogenesis are known. Therelevant T-cell and B-cell epitopes on type II collagen have beenidentified, and various immunological (e.g., delayed-typehypersensitivity and anti-collagen antibody) and inflammatory (e.g.,cytokines, chemokines, and matrix-degrading enzymes) parameters relatingto immune-mediated arthritis have been determined, and can thus be usedto assess test compound efficacy in the CIA model (Wooley, Curr. Opin.Rheum. 3:407-20, 1999; Williams et al., Immunol. 89:9784-788, 1992;Myers et al., Life Sci. 61:1861-78, 1997; and Wang et al., Immunol.92:8955-959, 1995).

The administration of anti-IL-21 antibodies to these CIA model mice isused to evaluate the use of anti-IL-21 antibodies to ameliorate symptomsand alter the course of disease. The addition of a second antibody to anantigen known to be involved in arthritis (or rheumatoid arthritis) canbe used. A bispecific antibody is particularly preferred if addition ofthe second antigen increases efficacy of the treatment and/or increasesspecificity of the antibody binding.

Inflammatory Bowel Disease (IBD)

In the United States approximately 500,000 people suffer frominflammatory bowel disease (IBD) which can affect either colon andrectum (ulcerative colitis) or both, small and large intestine (Crohn'sDisease). The pathogenesis of these diseases is unclear, but theyinvolve chronic inflammation of the affected tissues. Ulcerative colitis(UC) is an inflammatory disease of the large intestine, commonly calledthe colon, characterized by inflammation and ulceration of the mucosa orinnermost lining of the colon. This inflammation causes the colon toempty frequently, resulting in diarrhea. Symptoms include loosening ofthe stool and associated abdominal cramping, fever and weight loss.Although the exact cause of UC is unknown, recent research suggests thatthe body's natural defenses are operating against proteins in the bodywhich the immune systems thinks are “non-self” (an “autoimmunereaction”). Perhaps because they resemble bacterial proteins in the gut,these proteins may either instigate or stimulate the inflammatoryprocess that begins to destroy the lining of the colon. As the lining ofthe colon is destroyed, ulcers form releasing mucus, pus and blood. Thedisease usually begins in the rectal area and may eventually extendthrough the entire large bowel. Repeated episodes of inflammation leadto thickening of the wall of the intestine and rectum with scar tissue.Death of colon tissue or sepsis may occur with severe disease. Thesymptoms of ulcerative colitis vary in severity and their onset may begradual or sudden. Attacks may be provoked by many factors, includingrespiratory infections or stress.

Although there is currently no cure for UC available, treatments arefocused on suppressing the abnormal inflammatory process in the colonlining. Treatments including corticosteroids immunosuppressives (eg.azathioprine, mercaptopurine, and methotrexate) and aminosalicytates areavailable to treat the disease. However, the long-term use ofimmunosuppressives such as corticosteroids and azathioprine can resultin serious side effects including bone-thinning, cataracts, infection,and liver and bone marrow effects. In the patients in whom currenttherapies are not successful, surgery is an option. However, the surgeryinvolves the removal of the entire colon and the rectum.

There are several animal models that can partially mimic chroniculcerative colitis. The most widely used model is the2,4,6-trinitrobenesulfonic acid/ethanol (TNBS) induced colitis model,which induces chronic inflammation and ulceration in the colon. WhenTNBS is introduced into the colon of susceptible mice via intra-rectalinstillation, it induces T-cell mediated immune response in the colonicmucosa, in this case leading to a massive mucosal inflammationcharacterized by the dense infiltration of T-cells and macrophagesthroughout the entire wall of the large bowel. Moreover, thishistopathologic picture is accompanied by the clinical picture ofprogressive weight loss (wasting), bloody diarrhea, rectal prolapse, andlarge bowel wall thickening (Neurath et al. Intern. Rev. Immunol.19:51-62, 2000). Adoptive transfer of naive T cells into minorhistocompatibility mismatched or syngeneic immunocompromised mice leadsto development of colitis (Leach M W et al 1996, Powrie F et al, 1997)as well as skin lesions resembling psoriasis (Schon M P et al., Nat.Med. 2:183-8, 1997; Davenport C M et al., Int Immunopharmacol. 5:653-72,2002). Transplantation of as few as 0.2 million CD4+ CD25− T cells fromBALB/C or B10.D2 mice into immunocompromised C.B-17 SCID mice results inweight loss, hemoccult positive stool and development of skin lesions.The symptoms in these mice vary from colony to colony. This model ofcolitis/psoriasis has some similarities to human Crohn's disease andpsoriasis, and has been used extensively to test efficacy oftherapeutics for these diseases in humans.

Another colitis model uses dextran sulfate sodium (DSS), which inducesan acute colitis manifested by bloody diarrhea, weight loss, shorteningof the colon and mucosal ulceration with neutrophil infiltration.DSS-induced colitis is characterized histologically by infiltration ofinflammatory cells into the lamina propria, with lymphoid hyperplasia,focal crypt damage, and epithelial ulceration. These changes are thoughtto develop due to a toxic effect of DSS on the epithelium and byphagocytosis of lamina propria cells and production of TNF-alpha andIFN-gamma. Despite its common use, several issues regarding themechanisms of DSS-induced disease and its relevance to the human diseaseremain unresolved. DSS is regarded as a T cell-independent model becauseit is observed in T cell-deficient animals such as SCID mice.

The administration of anti-IL-21 antibodies to these TNBS, DSS or CD4+ Tcell-transfer models can be used to evaluate the use of IL-21antagonists to ameliorate symptoms and alter the course ofgastrointestinal disease. IL-21 may play a role in the inflammatoryresponse in colitis, and the neutralization of IL-21 activity byadministrating IL-21 antagonists is a potential therapeutic approach forIBD. The addition of a second antibody to an antigen known to beinvolved in inflammatory bowel disease can be used. A bispecificantibody is particularly preferred if addition of the second antigenincreases efficacy of the treatment and/or increases specificity of theantibody binding.

Psoriasis

Psoriasis is a chronic skin condition that affects more than sevenmillion Americans. Psoriasis occurs when new skin cells grow abnormally,resulting in inflamed, swollen, and scaly patches of skin where terminaldifferentiation of keratinocytes is altered. Plaque psoriasis, the mostcommon form, is characterized by inflamed patches of skin (“lesions”)topped with silvery white scales. Psoriasis may be limited to a fewplaques or involve moderate to extensive areas of skin, appearing mostcommonly on the scalp, knees, elbows and trunk. Although it is highlyvisible, psoriasis is not a contagious disease. The pathogenesis of thediseases involves T cell activation, altered antigen presentation andcytokine production by inflammatory dendritic cells, and chronicinflammation of the affected tissues. Anti-IL-21 antibodies of thepresent invention, could serve as a valuable therapeutic to reduceinflammation and pathological effects in psoriasis, other inflammatoryskin diseases, skin and mucosal allergies, and related diseases.

Psoriasis is a T-cell mediated inflammatory disorder of the skin thatcan cause considerable discomfort. It is a disease for which there is nocure and affects people of all ages. Psoriasis affects approximately twopercent of the populations of European and North America. Althoughindividuals with mild psoriasis can often control their disease withtopical agents, more than one million patients worldwide requireultraviolet light treatments or systemic immunosuppressive therapy.Unfortunately, the inconvenience and risks of ultraviolet radiation andthe toxicities of many therapies limit their long-term use. Moreover,patients usually have recurrence of psoriasis, and in some casesrebound, shortly after stopping immunosuppressive therapy. Anti-IL-21antibodies can be tested using a recently developed a model of psoriasisbased on the CD4+CD45RB transfer model (Davenport et al., Internat.Immunopharmacol., 2:653-672, 2002).

In addition to other disease models described herein, the activity ofanti-IL-21 antibodies on inflammatory tissue derived from humanpsoriatic lesions can be measured in vivo using a severe combined immunedeficient (SCID) based mouse model. Several mouse models have beendeveloped in which human cells or tissue grafts are implanted intoimmunodeficient mice (collectively referred to as xenograft models);see, for example, Caftan and Douglas, Leuk. Res. 18:513-22, 1994 andFlavell, Hematological Oncology 14:67-82, 1996. As an in vivo xenograftmodel for psoriasis, human psoriatic skin tissue is grafted onto SCIDmice, and the mice are subsequently challenged with an appropriateantagonist. Moreover, other psoriasis animal models in the art may beused to evaluate IL-21 antagonists, such as human psoriatic skin graftsimplanted into the AGR129 mouse model, and challenged with anappropriate antagonist (e.g., see, Boyman et al., J. Exp. Med. Onlinepublication #20031482, 2004). Similarly, tissues or cells derived fromhuman colitis, IBD, arthritis, or other inflammatory lesions can be usedin the SCID model to assess the anti-inflammatory properties of theanti-IL-21 antibodies described herein.

Efficacy of treatment is measured and statistically evaluated asincreased anti-inflammatory effect within the treated population overtime using methods well known in the art. Some exemplary methodsinclude, but are not limited to measuring for example, in a psoriasismodel, epidermal thickness, the number of inflammatory cells in theupper dermis, and the grades of parakeratosis. Such methods are known inthe art and described herein. For example, see Zeigler et al., LabInvest 81:1253, 2001; Zollner et al., J. Clin. Invest. 109:671, 2002;Yamanaka et al., Microbiol. Immunol. 45:507, 2001; Raychaudhuri et al.,Br. J. Dermatol. 144:931, 2001; Boehncke et al., Arch. Dermatol. Res.291:104, 1999; Boehncke et al., J. Invest. Dermatol. 116:596, 2001;Nickoloff et al., Am. J. Pathol. 146:580, 1995; Boehncke et al., J.Cutan. Pathol. 24:1, 1997; Sugai et al., J. Dermatol. Sci. 17:85, 1998;and Villadsen et al., J. Clin. Invest. 112:1571, 2003. Inflammation mayalso be monitored over time using well-known methods such as flowcytometry (or PCR) to quantitate the number of inflammatory or lesionalcells present in a sample, score (weight loss, diarrhea, rectalbleeding, colon length) for IBD, paw disease score and inflammationscore for CIA RA model.

The administration of anti-IL-21 antibodies to these psoriasis modelmice is used to evaluate the use of anti-IL-21 antibodies to amelioratesymptoms and alter the course of disease. The addition of a secondantibody to an antigen known to be involved in psoriasis can be used. Abispecific antibody is particularly preferred if addition of the secondantigen increases efficacy of the treatment and/or increases specificityof the antibody binding.

Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is an immune-complex related disordercharacterized by chronic IgG antibody production directed at ubiquitousself antigens (e.g. anti-dsDNA). The effects of SLE are systemic, ratherthan localized to a specific organ, although glomerulonephritis mayresult in some cases (i.e. lupus nephritis). Multiple chromosomal locihave been associated with the disease and may contribute towardsdifferent aspects of the disease, such as anti-dsDNA antibodies andglomerulonephritis. CD4+ T cells have been shown to play an active partin mouse models of SLE (Horwitz, Lupus 10:319-320, 2001; Yellin andThienel, Curr. Rheumatol. Rep., 2:24-37, 2000). The role for CD8+ Tcells is not clearly defined, but there is evidence to suggest that“suppressor” CD8+ T cell function is impaired in lupus patients (Filaciet al., J. Immunol., 166:6452-6457, 2001; Sakane et al, J. Immunol.,137:3809-3813, 1986).

IL-21 has been convincingly shown to induce the differentiation of naïvehuman B cells into antibody-secreting plasma cells (Ozaki et al., J.Immunol. 173:5361, 2004; Ettinger et al., J. Immunol. 175:7867-79, 2005;Ettinger et al, J. Immunol. 178:2872-82, 2007; Kuchen et al. J Immunol.179:5886-96, 2007). Ozaki et al., (J. Immunol. 173:5361, 2004) alsodemonstrated that IL-21 expression is elevated in lupus-prone BXSB-Yaamice, a model for SLE, at an age when the early characteristics ofautoimmune processes first become evident. Treatment of these BXSB-Yaamice with a murine IL-21 antagonist partially inhibits various diseaseparameters, including glomerulonephritis (Bubier et al., Ann N Y AcadSci. 1110:590-601, 2007). The same IL-21 antagonist has also been shownto be efficacious in another pre-clinical disease model of SLE, theMRL/lpr mouse (Herber et al. J. Immunol. 178: 3822-3830, 2007).Moreover, because IL-21 limits development of Treg cells, administrationof anti-IL-21 antibodies could provide a more robust T cell suppressorfunction in lupus patients where that function is compromised (Lamprechtet al. Blood. 112(8):3339-47, 2008).

Data obtained from 24 SLE patients and 15 healthy controls showedthat 1) IL-21 mRNA expression is significantly increased in CD4+ T cellsfrom lupus patients compared to controls, 2) IL-21 levels aresignificantly elevated in sera from patients with active compared toinactive SLE or controls, as determined using a commercial IL-21 ELISAkit (Invitrogen, Carlsbad, Calif.), 3) IL-21 enhances CD4+ T cells andCD19+ B cells proliferation in patients and controls in a dose dependentfashion, 4) IL-21 enhances anti-CD40 induced plasma cell differentiationin normal controls and SLE patients, and 5) elevated levels of IL-21 maycontribute to proliferation of autoreactive CD4+ T cells and plasma celldifferentiation in SLE ((Rus, V., ACR Presentation #1760, 2008 AmericanCollege of Rheumatology meeting, Oct. 24-29, 2008).

Anti-IL-21 antibodies can be administered in combination with otheragents already in use in autoimmunity including immune modulators suchas IFNγ, NOVANTRONE®, ENBREL®, BETAFERON®, REMICADE®, LEUKINE® andPROLEUKIN®. Anti-IL-21 antibodies can be administered in combinationwith other agents already in use in the cancer therapy of multiplemyeloma, Hodgkin's Lymphoma or acute T-cell leukemia such as THALOMID®or with steroids such as dexamethasone or prednisone. Establishing theoptimal dose level and scheduling for anti-IL-21 antibodies is done by avariety of means, including study of the pharmacokinetics andpharmacodynamics of anti-IL-21 antibodies; determination of effectivedoses in animal models, and evaluation of the toxicity of anti-IL-21antibodies. Direct pharmacokinetic measurements done in primates andclinical trials can then be used to predict theoretical doses inpatients that achieve plasma anti-IL-21 antibody levels that are ofsufficient magnitude and duration to achieve a biological response inpatients. The addition of a second antibody to an antigen known to beinvolved in SLE can be used. A bispecific antibody is particularlypreferred if addition of the second antigen increases efficacy of thetreatment and/or increases specificity of the antibody binding.

Transplant Rejection

Recipients of transplanted solid organs may develop acute or chronicrejection of the allograft due to histocompatability mismatch. Thegeneration of antibodies directed against the HLA molecules(alloantibodies) in these patients results from presentation of theforeign antigen to T cells. Alloantibodies may mediate tissue damage inthe graft through formation of immune complexes, complement fixation,and antibody mediated cellular cytotoxicity directed by boundalloantibodies. The complement cascade also releases local factors thatactivate endothelial cells and cause vasculopathy within the graft. Thecomplement product C4d is an early marker in both acute and chronictransplant rejection, and can be detected in sub-clinical cases prior toovert pathological changes (Racusen and Haas, Clin J Am Soc Nephrol 1:415-420, 2006; Moll and Pascual, Am J Transplantation 5: 2611-2618,2005; Tinkam and Chandraker, Clin J Am Soc Nephrol 1: 404-414, 2006).Patients are screened for anti-HLA alloantibodies (panel reactiveantibody) prior to transplantation. Patients may be highly sensitizeddue to prior allograft failure, blood transfusions, or multiplepregnancies. The presence of alloantibodies in highly sensitizedtransplant patients complicates their care, as increasedimmunosuppressive therapies may be required and the chance of acuterejection is high Maid et al., Curr Opin Immunol 13:577-581, 2001). Insome cases, B cell targeting agents (mycophenolic acid or rituximab) areused, although this therapeutic strategy does not directly target theantibody-secreting plasma cells. Plasmapheresis is also used to reducecirculating immunoglobulin. In all cases, transplant recipients aretreated with T cell targeted immunosuppressive agents to reduce the riskof rejection, and may be slowly “weaned” from these regimens astolerance to the graft is established (Seyfort-Margolis and Turka, JClin Invest 118(8): 2684-2685, 2008; Taylor et al., Crit Rev OncolHematol 56:23-46, 2005; Amante and Ejercito, Transplant Proc 40:2274-2280, 2008).

Development of antibody secreting plasma cells requires cognate helpfrom CD4 T cells in addition to the specialized microenvironments thatsupport plasma cell survival (Tarlington et al., Curr Opin Immunol20:162-169, 2008). Cytokine secretion by activated T cells is necessaryfor differentiation and survival of plasma cells, and is known to affectthe nature of the antibody response and Ig isotype. Models exist tomonitor T cell dependent antibody responses in murine and non-humanprimate species. These methods are well understood by those skilled inthe art. Kinetics and magnitude of primary or secondary antibodyresponses against model peptide antigens such as ovalbumin, tetanustoxoid, sheep red blood cells, or trinitrophenyl modified keyhole limpethemocyanin are monitored using assays that detect total orantigen-specific antibodies, including IgG sub-types, IgM, IgE, or IgAin serum of treated animals. In some models, affinity maturation of theantibodies can also be monitored. These models may be used to test theeffects of therapeutic drugs that alter B cell help by T cells and thatblock cytokines thought to be important for plasma cell differentiationand survival.

Studies of allograft rejection are conducted in many animal species. Forexample, a renal transplant model in cynomolgus monkeys may representthe effects of chronic alloantibody mediated renal allograft rejectionin humans. Allograft tolerizing regimens are performed prior totransplant in some cases. The presence of donor-specific alloantibodiesis monitored by flow cytometric analysis of recipient serum withmismatched peripheral blood leukocytes, and deposition of the complementproduct C4d is detected in biopsies from the renal allograft (Smith etal., Am J Transplant 6:1790-1798, 2006; Smith et al., Am J Transplant8:1-11, 2008). Acute and chronic transplant models may be conducted inmurine species by those skilled in the art.

Administration of anti-IL-21 antibodies in a model of T cell dependentantibody response or a model of allograft rejection is used to evaluatethe clinical utility of anti-IL-21 antibodies to reduce alloantibodyresponses, and ameliorate symptoms of allograft rejection, or as part ofthe pre-transplant therapeutic or tolerizing regimen for transplantrecipients, including highly sensitized transplant patients. Theaddition of a second antibody to an antigen known to be involved intransplant rejection can be used. A bispecific antibody is particularlypreferred if addition of the second antigen increases efficacy of thetreatment and/or increases specificity of the antibody binding.

The invention is further illustrated by the following non-limitingexamples.

EXAMPLES Example 1 Preparation of IL-21 Proteins and Antibodies

A. Immunizations and Hybridomas

IL-21 protein was produced as described in U.S. Pat. No. 7,250,274,incorporated in its entirety herein. Soluble IL-21 receptor proteinswere produced as described in U.S. Patent Application 2007-0122413 andU.S. Pat. No. 6,777,539, both incorporated in their entirety herein.Anti-IL-21 monoclonal antibodies were produced in wild type micegenerating murine antibodies and in transgenic mice generating fullyhuman antibodies (Medarex, Princeton, N.J.). Mice were immunized withhuman IL-21 protein. Briefly, the mice were initially immunized bysubcutaneous injection with 30 μg of purified recombinant IL-21(produced in E. coli at ZymoGenetics) conjugated with BSA (ImjectPharmalink Immunogen Kit, Pierce) and administered in combination withCpG (oligonucleotide murine TLR9 ligand and GM-CSF (GranulocyteMacrophage Colony Stimulatory Factor, R&D, Minneapolis, Minn.) andEmulsigen®-P adjuvant (MVP Laboratories, INC, Omaha, Nebr.) as permanufacturer's instructions. Following the initial immunization, each ofthe mice received three additional 30 μg of IL-21 in Emulsigen®-Padjuvant via the subcutaneous route in weekly intervals. Seven daysafter the fourth immunization, the mice were bled via the retro orbitalplexus and the serum separated from the blood for analysis of itsability to bind to IL-21.

Splenocytes were harvested and pooled from two high-titer BALB/c mice ortransgenic mice and fused to P3-X63-Ag8.653 mouse myeloma cells usingPEG 1450 in a single fusion procedure (2:1 fusion ratio, splenocytes tomyeloma cells, “Antibodies: A Laboratory Manual”, E. Harlow and D. Lane,Cold Spring Harbor Press). Following 9 days growth post-fusion, specificantibody-producing hybridoma pools were identified by Direct and CaptureELISA using recombinant IL-21 protein, untagged and human IgG Fc tagged,as specific antibody target. Positive hybridoma pools were analyzedfurther for their ability to block the Ligand to receptor binding, whichis measured as the level of STAT3-phosphorylation followingligand-receptor interaction (“phosphor-STAT3 neutralization assay”) ofpurified recombinant IL-21 protein on BaF3 cells expressing the IL-21receptor sequence. Monoclonal antibodies purified from tissue culturemedia were characterized for their ability to block the ligand-receptorinteraction (“phosphor-STAT3 neutralization assay”) of purifiedrecombinant IL-21 on Baf3 cells expressing the receptor sequences.“Neutralizing” monoclonal antibodies were identified in this manner.

Hybridoma pools yielding positive results by the “phosphor-STAT3neutralization assay” and ELISA formats were cloned at least two timesby limiting dilution. In these assays, samples were titrated usingstandard low density dilution (less than one cell per well) to see whichclone will maintain the highest OD reading. Using the results from boththe neutralization and titration assays, two specific clones from eachinitial master well were selected for further analysis. These aresubjected to an additional round of cloning to ensure culturehomogeneity and screened using the Direct ELISA. After one additionaltitration assay, two final IL-21 clones were selected. Hybridoma cloneswere cultured in a growth medium of 90% Iscove's Modified Dulbecco'smedium with 2 mM L-glutamine, 100 μg/mL penicillin, and 100 μg/mLstreptomycin sulfate, and 10% Fetal Clone I Serum (HycloneLaboratories). The clones were propagated by seeding cultures at 2×10⁵cells/ml and maintaining between 1×10⁵ and 5×10⁵ cell/ml at 37° C. and5-6% CO₂. Cells were adapted to serum free conditions upon subsequenttransfers. Cells are frozen in 90% serum, 10% DMSO and stored in vaporphase of a liquid nitrogen freezer.

The purified monoclonal antibodies produced by the hybridoma clones werecharacterized in a number of ways including binning (i.e, determining ifeach antibody could inhibit the binding of any other antibody), epitopemapping using peptides, relative affinity, and neutralization.

Methods for producing heterologous antibodies from transgenic mice areknown, see for example, Lonberg, Nat. Biotech. 23(9):1117-25, 2005;Tomizuka et al. PNAS 97(2):722-727, 2000; and U.S. Pat. No. 5,625,126.

The following hybridomas producing murine anti-human IL-21 monoclonalantibodies have been deposited with the American Type CultureCollection, Manassas, Va. clone 338.5.4 ATCC No. (PTA-8317), clone338.11.5 ATCC No. (PTA-8314), clone 338.14.3 ATCC No. (PTA-8313), clone338.15.5 ATCC No. (PTA-8315), clone 338.17.3 ATCC No. (PTA-8316), clone338.24.5 ATCC No. (PTA-8430), clone 338.25.6 ATCC No. (PTA-8431), clone338.39.5 ATCC No. (PTA-8432), clone 338.29.2 ATCC No. (PTA-8433), clone338.28.6 ATCC No. (PTA-8434).

The following hybridomas producing human anti-human IL-21 monoclonalantibodies have been deposited with the American Type CultureCollection, 10801 University Boulevard, Manassas, Va., 20110-2209. Table1 provides complete amino acid sequences for the variable heavy (VH) andvariable light (VL) chains of the antibodies. Also included aresequences for CDR1, CDR2 and CDR3 of VH and VL regions for eachantibody. The corresponding nucleotide sequences are found in thesequence listing. Included in the deposit, but not in Table 1, is ahybridoma designated 366.345.6.11, ATCC No. PTA-8788.

TABLE 1 Clone ATCC VH VH VH VL VL VL Complete Number No. CDR1 CDR2 CDR3CDR1 CDR2 CDR3 Sequence 362.75.1. PTA- SRTYR SIYYRG QSGYS RASQS DASNRQQRSN

1.7 8791 WG STFYNP GYDWF VSSFLA AT WITMKHLWFFLLLVAAPRWVLSQLQLQESGPGLVKPSETLSL SEQ ID SLKS DP SEQ ID SEQ IDSEQ ID TCTVSGGSISSRTYRWGWIRQPPGKELEWIGSIYYRGST NO: 15 SEQ ID SEQ IDNO: 23 NO: 25 NO: 27 FYNPSLKSRVTVSVDTSKNQFSLKLSSVTAADTAVYYCA NO: 17NO: 19 RQSGYSGYDWFDPWGQGTLVTVSS SEQ ID NO: 13

MEAPAQLLFLLLLWLPDTTGEIVLTQSPATLSLSPGERATLSCRASQSVSSFLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWITF GQGTRLEIK SEQ ID NO: 21362.78.1. PTA- SYGMH FIWYDG DGDSSD RASQSV GASSRA QQYGSW

44 8790 SEQ ID SDKYYA WYGDYY SSSYLA T TMEFGLSWVFLVALLRGVQCQVQLVESGGGVVQPGRSLRL NO: 31 DSVKG FGMDV SEQ ID SEQ IDSEQ ID SCAASGFTFSSYGMHWVRQAPGKGLEWVAFIWYDGSDKY SEQ ID SEQ ID NO: 39NO: 41 NO: 43 YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR NO: 33 NO: 35DGDSSDWYGDYYFGMDVWGQGTTVTVSS SEQ ID NO: 29

METPAQLLFLLLLWLPDTTGEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSWTF GQGTKVEIK SEQ ID NO: 37 362.597.PTA- TYGMH FIWYDG DGDSSD RASQSV GASSRA QQYGSW

3.15 8786 SEQ ID SDKYYA WYGDYY SSSYLA T TMEFGLSWVFLVALLRGVQCQVQLVESGGGVVQPGRSLRL NO: 47 DSVKG FGMDV SEQ ID SEQ IDSEQ ID SCAASGFTFSTYGMHWVRQAPGKGLEWVAFIWYDGSDKY SEQ ID SEQ ID NO: 55NO: 57 NO: 59 YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR NO: 49 NO: 51DGDSSDWYGDYYFGMDVWGQGTTVTVSS SEQ ID NO: 45

METPAQLLFLLLLWLPDTTGEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSWTF GQGTKVEIK SEQ ID NO: 53 366.328.PTA- SYSMN SITSGS ERGWGY RASQDI DASSLE QQFNSY

10.63 8789 SEQ ID YYIHYA YGMDV DSALA S PYTMELGLRWVFLVAILEGVQCEVQLVESGGGLVKPGGSLRL NO: 63 DSVKG SEQ ID SEQ IDSEQ ID SEQ ID SCAASGFIFSSYSMNWVRQAPGKGLEWVSSITSGSYYIH SEQ ID NO: 67NO: 71 NO: 73 NO: 75 YADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCVR NO: 65ERGWGYYGMDVWGQGTTVTVSS SEQ ID NO: 61

MDMRVPAQLLGLLLLWLPGARCAIQLTQSPSSLSASVGDRVTITCRASQDIDSALAWYQQKPGKAPKILIHDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYP YTFGQGTKLEIK SEQ ID NO: 69366.552. PTA- SDFWG YISSRG SAGVTD RASQGI VASSLQ QQANSF

11.31 8787 SEQ ID STNYN- FDF SSWLA S PLTMKHLWFFLLLVAAPRWVLSQVQLQESGPGLVKPSETLSL NO: 79 PSLKR SEQ ID SEQ IDSEQ ID SEQ ID TCTVSGGSISSDFWGWIRQPPGKGLEWIGYISSRGSTNY SEQ ID NO: 83NO: 87 NO: 89 NO: 91 NPSLKRRVTISVDTSRNQFSLKLSSVTAADTAVYYCARS NO: 81AGVTDFDFWGQGTLVTVSS SEQ ID NO: 77

MDMMVPAQLLGLLLLWFPGSRCDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQHKPGKAPKLLIYVASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATTYCQQANSFP LTFGGGTKVEIK SEQ ID NO: 851B: Expression of Clone 362.78.1.44 Immunoglobulin Heavy and Light ChainGenes in a Mammalian Cell Line to Produce 362.78-CHO

Human anti-human IL-21 monoclonal antibody (derived from hybridoma clone362.78.1.44) was expressed in CHO cells using two expression cassettes.The VH chain was fused to a modified human IgG1 constant region. Themodified IgG1, IgG1.1, contained five amino acid substitutions to reduceeffector functions. The human anti-human IL-21 heavy chain was linked toa dihydrofolate reductase (DHFR) selectable marker with an internalribosomal entry site (IRES) sequence. The expression of the humananti-human IL-21 heavy chain and the DHFR selectable marker weredirected by a constitutive synthetic promoter consisting of a fusion ofthe human cytomegalovirus (CMV) enhancer and the myeloproliferativesarcoma virus (MPSV) enhancer/promoter. The simian virus 40 (SV40)polyadenylation signal was used to terminate transcription at the end ofthe DHFR selectable marker. The VL chain was fused to the humanimmunoglobulin kappa constant region. The human anti-human IL-21 lightchain was linked to a puromycin resistance (puroR) selectable markerwith an IRES sequence. The expression of the human anti-human IL-21light chain and the puroR selectable marker were directed by aconstitutive synthetic promoter consisting of a fusion of the human CMVenhancer and the MPSV enhancer/promoter. The SV40 polyadenylation signalwas used to terminate transcription at the end of the puroR selectablemarker. The human anti-human IL-21 heavy and light chain expressioncassettes were co-transfected into CHO DXB-11 host cells. Puromycinselection was followed by methotrexate selection to obtain high, stableexpression of human anti-human IL-21 monoclonal antibody. TheCHO-expressed version of IL-21 mAb clone 362.78.1.44 will be referred toin subsequent Examples as “362.78-CHO.”

Example 2 Anti-IL-21 Monoclonal Antibodies Bind Human IL-21 Proteins andPeptides

2A. Binding and Neutralization of Peptides

The ability of the anti-human IL-21 binding and neutralizing monoclonalantibodies to bind to human IL-21, mutant human IL-21 protein, and humanIL-21 sequence derived synthetic peptides was demonstrated in the DirectELISA assay format.

The following peptides were used:

peptide #1 ((SEQ ID NO:3) pyroGlu GQDRHMIRMRQLIDIVDQLKC;

peptide #2 ((SEQ ID NO: 4) NDLVPEFLPAPEDVETNC,

peptide #3 ((SEQ ID NO: 5) NVSIKKLKRKPPSTNAGRRQKHRLTC,

peptide #4 ((SEQ ID NO:6) CDSYEKKPPKEFLERFKSLLQKMIHQHLS and

Recombinant human IL-21 (SEQ ID NO: 2), human IL-21 sequence derivedsynthetic peptides, and recombinant mutant human IL-21 (SEQ ID NO: 7)were separately immobilized onto the surface of 96 well polystyreneELISA plates in a volume of 100 μL/well at a concentration of 1 μg/mL inCoating Buffer (0.1M Na₂CO₃, pH 9.6). Plates were incubated overnight at4° C. after which unbound protein was aspirated and the plates washedtwice with 300 μL/well of Wash Buffer (PBS-Tween defined as 0.137M NaCl,0.0022M KCl, 0.0067M Na₂HPO₄, 0.0020M KH₂PO₄, 0.05% v/w polysorbate 20,pH 7.2). Wells were blocked with 200 μL/well of Blocking Buffer(PBS-Tween plus 1% w/v bovine serum albumin (BSA)) for 1 hour, afterwhich the plates were washed twice with Wash Buffer. Antibody dilutionswere prepared in 5% fetal bovine serum (FBS)/Iscove's ModifiedDulbecco's Media (IMDM) medium and adjusted to 1 ug/ml. Duplicatesamples of each antibody dilution were then transferred to the assayplates, 100 μL/well, in order to bind anti-human IL-21 proteins.Following 1 hour incubation at ambient temperature, the wells wereaspirated and the plates washed twice as described above. Horseradishperoxidase (HRP) labeled Goat anti Mouse IgG, Fc specific or Goat antiRat IgG, Fc specific, or Goat anti Human IgG, Fc specific (JacksonImmunoResearch Laboratories, West Grove, Pa.) at a dilution of 1:5000with 5% FBS/IMDM medium was then added to each well, 100 μL/well, andthe plates incubated at ambient temperature for 1 hour. After removal ofunbound HRP conjugated antibody, the plates were washed five times, 100μL/well of tetra methyl benzidine (TMB) (BioFX Laboratories, OwingsMills, Md.) added to each well and the plates incubated for 3 minutes atambient temperature. Color development was stopped by the addition of100 μL/well of 450 nm TMB Stop Reagent (BioFX Laboratories, OwingsMills, Md.) and the absorbance values of the wells read on a MolecularDevices Spectra MAX 340 instrument at 450 nm.

TABLE 2 Monoclonal mouse anti-human IL-21 antibody reactivity to humanIL-21 protein, mutant human IL-21 protein and human IL-21 sequencederived peptides Mouse Anti- Binding (B) human Neutralizing PurifiedIL-21 Ab (N) Ab Peptide Peptide Peptide Peptide IL-21 Clone # IsotypeLot# IL-21 1 2 3 4 mutant 338.5.4 B/N E10274 +/− 0 0 0 0 +/− IgG1338.11.5 B E10276 +++ 0 +++ 0 0 +++ IgG1 338.14.3 B E10273 +/− 0 0 0 0 0IgG1 338.15.5 B E10275 +++ 0 0 0 +++ 0 IgG1 338.28.6 B E10329 +++ 0 0 00 + IgG1 338.39.5 B E10330 +++ 0 0 +++ 0 +++ IgG1 Reactivity: None (0)Weak (+) Moderate (++) Strong (+++)

TABLE 3 Monoclonal human anti-human IL-21 antibody reactivity to humanIL-21 protein, mutant human IL-21 protein and human IL-21 sequencederived peptides Human Anti- Binding (B) human IL-21 NeutralizingPurified Peptide 1 Ab (N) Ab N- Peptide 4 IL-21 Clone # Isotype Lot#IL-21 Term Peptide 2 Peptide 3 C-Term mutant 362.75.1.1 B/N E10364 +++ 00 0 +++ 0 IgG 362.78.1.44 B/N E10554 ++ 0 0 0 0 + IgG 362.597.3 B/NE10366 +++ 0 0 0 0 ++ IgG 366.328.10 B/N E10416 +++ 0 0 0 0 +++ IgG366.345.6.11 B E10476 +++ 0 0 0 0 ++ IgG 366.552.11 B/N E10435 +++ 0 0 0++ 0 IgG Reactivity: None (0) Weak (+) Moderate (++) Strong (+++)2B Measurement of the Binding Affinities of Anti-Human IL-21 MonoclonalAntibody 362.78-CHO to Human IL-21 and Cynomolgus Monkey IL-21 bySurface Plasmon Resonance (Biacore)

The anti-IL-21 monoclonal antibody 362.78-CHO was evaluated for itsbinding affinity to human recombinant IL-21 and cynomolgus recombinantIL-21 using surface plasmon resonance.

Affinity Determination: Kinetic rate constants and equilibriumdissociation constants were measured for the interaction of theanti-human IL-21 monoclonal antibody 362.78-CHO with human IL-21 andcynomolgus IL-21 via surface plasmon resonance. The association rateconstant (k_(a) (M⁻¹s⁻¹)) is a value that reflects the rate of theantigen-antibody complex formation. The dissociation rate constant(k_(d) (s⁻¹)) is a value that reflects the stability of this complex. Bydividing the dissociation rate constant by the association rate constant(k_(d)/k_(a)) the equilibrium dissociation constant (K_(D) (M)) isobtained. This value describes the binding affinity of the interaction.Antibodies with similar K_(D) can have widely variable association anddissociation rate constants. Consequently, measuring both the k_(a) andk_(d) of antibodies helps to more uniquely describe the affinity of theantibody-antigen interaction.

Materials and Methods: Binding kinetics and affinity studies wereperformed on a Biacore T100™ system (GE Healthcare, Piscataway, N.J.).Methods for the Biacore T100™ were programmed using BIACORE T100™Control Software, v 1.1.1. For these experiments, the 362.78-CHOantibody was either captured onto a CM4 sensor chip via a goatanti-human IgG Fc-gamma antibody (Jackson ImmunoResearch, West Grove,Pa.), or it was minimally biotinylated with a 1:100 mass ratio ofSulfo-NHS-LC-Biotin (Pierce, Rockford, Ill.) in a buffer of PBS pH 7.4then captured onto a streptavidin (SA) chip. All binding experimentswere performed at 25° C. in a buffer of 10 mM HEPES, 300 mM NaCl, 5 mMCaCl₂, 0.05% Surfactant P20 (Biacore), 1 mg/mL bovine serum albumin, pH8.0.

For the experiments with the goat anti-human IgG Fc-gamma antibody, thecapture antibody was diluted to concentration of 50 μg/mL in 10 mMsodium acetate pH 5.0, and then covalently immobilized to all four flowcells of a CM4 sensor chip using amine coupling chemistry (EDC:NHS).After immobilization of the antibody, the remaining active sites on theflow cell were blocked with 1 M ethanolamine. A capture antibody densityof approximately 3500 RU was obtained. The anti-IL-21 antibody362.78-CHO was captured onto flow cell 2, 3, and 4 of the CM4 chip atthree different densities (ranging from 25 to 150 RU). Capture of the362.78-CHO antibody to the immobilized surface was performed at a flowrate of 10 μL/min. The Biacore instrument measures the mass of proteinbound to the sensor chip surface, and thus, capture of the test antibodywas verified for each cycle. Serial dilutions of human recombinant IL-21or cynomolgus recombinant IL-21 (ZymoGenetics) were prepared from 40nM-0.003 nM (1:5 serial dilutions). The serial dilutions were injectedover the surface and allowed to specifically bind to the 362.78-CHOantibody captured on the sensor chip. Duplicate injections of each IL-21antigen concentration were performed with an association time of either6.5 or 7 minutes and dissociation time of either 10, 15, or 60 minutes.Kinetic binding studies were performed with a flow rate of 50 μL/min. Inbetween cycles, the flow cell was washed with 20 mM hydrochloric acid toregenerate the surface. This wash step removed both the captured testantibody and any bound antigen from the immobilized antibody surface.The 362.78-CHO antibody was subsequently captured again in the nextcycle.

For the experiments with the minimally biotinylated 362.78-CHO, thebiotinylated antibody was captured onto flow cell 2, 3, and 4 of the SAchip at three different densities (ranging from 150 to 1200 RU). Captureof the biotinylated-362.78-CHO antibody to the surface was performed ata flow rate of 10 μL/min. Serial dilutions of human recombinant IL-21 orcynomolgus recombinant IL-21 (ZymoGenetics) were prepared either from 50nM-0.001 nM (1:4 serial dilutions) or from 40 nM-0.003 nM (1:5 serialdilutions). These serial dilutions were injected over the surface andallowed to specifically bind to the 362.78-CHO antibody captured on thesensor chip. Duplicate injections of each IL-21 antigen concentrationwere performed with an association time of either 6.5 or 7 minutes anddissociation time of either 10, 15, or 60 minutes. Kinetic bindingstudies were performed with a flow rate of 50 μL/min. In between cycles,the flow cell was washed with 20 nM hydrochloric acid to regenerate thesurface. This wash step removed any bound antigen from the immobilizedantibody surface. The wash cycle did not remove the biotinylated362.78-CHO antibody from the sensor surface, and the antibody wassubsequently available to bind the next antigen sample.

Data was compiled using the BIACORE T100™ Evaluation software (version1.1.1). Data was processed by subtracting reference flow cell and blankinjections. Baseline stability was assessed to ensure that theregeneration step provided a consistent binding surface throughout thesequence of injections. Duplicate injection curves were checked forreproducibility. Based on the binding of the monovalent IL-21 to abivalent antibody, the 1:1 binding interaction model was determined tobe appropriate. The reference-subtracted binding curves from three flowcells (FC2-1, FC3-1, FC4-1) were globally fit to the 1:1 binding modelwith a multiple Rmax and with the RI set to zero. The data fit well tothe 1:1 binding model with good agreement between the experimental andtheoretical binding curves. The chi² and standard errors associated thefits were low. There was no trending in the residuals.

Results: For the interaction of 362.78-CHO with human IL-21, data wascompiled from four separate experiments. The k_(a) of the multipleexperiments ranged from 3E+07 to 5+07 (M⁻¹ s⁻¹), while the k_(d) rangedfrom 3E-06 to 3E-05 (s⁻¹). The calculated K_(D) ranged from 0.9E-13 to8E-13 (M).

For the interaction of 362.78-CHO with cynomolgus IL-21, data wascompiled from three separate experiments. The k_(a) was 3E+07 (M⁻¹s⁻¹)for each experiment, while the k_(d) ranged from 2E-04 to 5E-04 (s⁻¹).The calculated K_(D) ranged from 0.9E-11 to 2E-11 (M).

Example 3 Species Cross Reactivity Experiments

Determination of ability of anti-human IL-21 antibodies to cross-reactand bind murine or cynomolgous monkey IL-21 protein or human IL-21sequence derived synthetic peptides

Species cross-reactivity studies can be important to demonstratespecificity for therapeutic antagonist development strategies. In orderto determine whether the anti-human IL-21 binding and neutralizingentities described herein may cross-react and bind to murine orcynomolgus IL-21 (and therefore, justify the cynomolgus monkey or mouseas a viable test species), it was necessary to demonstrate comparablebinding of the antibodies to recombinant human, murine and cynomolgousmonkey IL-21 in the various assay formats. One of the methods fortesting binding of the monoclonal antibodies is by their performance inimmunoblot (Western blot) assays. Recombinant human IL-21 (SEQ ID NO:2),recombinant murine IL-21 (SEQ ID NO:11), recombinant cynomolgus IL-21(SEQ ID NO:9), human IL-21 sequence derived synthetic peptides: peptide#1 pyr30-K50 (Seq ID 3), peptide #2 N54-C71 (Seq ID NO:4), peptide #3N97-C122 (Seq ID NO:5), peptide #4 C125-S153 (Seq ID NO:6) conjugated toovalbumin, or an irrelevant control cytokine, recombinant human IFN-λ(ZymoGenetics) were submitted to sodium dodecyl sulfate-polyacrylamidegel electrophoresis (SDS-PAGE) using 4-12% BisTris polyacrylamide gels(Invitrogen, Inc.) and transferred to nitrocellulose membranes usingstandard methods and a buffer containing 25 mM Tris, 186 mM glycine and40% methanol.

For Western blots, the non-specific sites on the membranes were blockedwith a buffer containing 20 mM Tris, pH 7.4, 0.5 mM EDTA, 0.5% IGEPALCA-630, 150 mM NaCl, 0.25% gelatin, and 1% casein hydrolysate blockingsolution (Western Blocking Reagent, Roche Diagnostics, Inc., BaselSwiterzerland) (Blocking Buffer). The membranes were then incubated for2 hrs at room temperature with purified monoclonal antibody (10 ng/ml or100 ng/ml) in the Blocking Buffer followed by a 2 hr incubation withperoxidase conjugated donkey anti-human IgG (Jackson Laboratories, BarHarbor, Me.). The membranes were washed 5 times with theTris/EDTA/IGEPAL/NaCl/gelatin Blocking buffer which lacked the caseinhydrolysate and developed with SUPERSIGNAL™ DuraWestLuminol/Enhancer/Peroxidase Solution (Pierce, Rockford, Ill.) forchemoluminescence detection. The blots were visualized using X-ray filmbased standard methods.

TABLE 4 Monoclonal Human Anti-Human IL-21 Antibody Reactivity in WesternBlot Analysis +Hu IL-21 +Hu IL-21 +Hu IL-21 +Hu IL-21 Hu Cyno Mu PeptidePeptide Peptide Peptide Clone* IL-21 IL-21 IL-21 A1744 A1750 A1751 A1752362.35.1.2 0 0 0 0 0 0 0 362.37.3 +++ +++ +++ 0 0 0 0 362.75.1.1 +++ ++++++ 0 0 0 +++ 362.78.1 ++ ++ 0 0 0 0 0 362.108.1.2 0 0 0 0 0 0 0362.172.2 +++ +++ +++ 0 0 0 0 362.216.2 0 +/− 0 0 0 0 0 362.256.1 0 0 00 0 0 0 362.303.1.1 0 0 0 0 0 0 0 362.378.1 +++ +++ +++ 0 0 0 +362.468.3 +/− + 0 0 0 0 0 362.564.1.4 ++ ++ 0 0 0 0 0 362.597.3 ++ +++/− 0 0 0 0 362.632.2 +++ +++ +++ 0 0 0 +++ 366.328.10 + + 0 0 0 0 0366.342.8 +++ +++ +++ 0 0 0 0 366.345.6.11 0 0 0 0 0 0 0 366.353.11.12 +++ 0 0 0 0 0 366.398.36 +++ +++ +++ 0 0 0 0 366.453.30 +++ +++ +++ 0 0 00 366.462.24.10 +++ +++ +++ 0 0 0 0 366.479.13 +++ +++ +++ 0 0 0 0366.552.11 +++ +++ +++ 0 0 0 0 366.565.7 0 0 0 0 0 0 0 366.617.7 +++ ++++++ 0 0 0 0 366.618.20 ++ ++ + 0 0 0 0 367.752.5 +++ +++ +++ 0 0 0 0368.626.24 +++ +++ ++ 0 0 0 0 Reactivity: None (0) Weak (+) Moderate(++) Strong (+++) *No Signals Observed with IFN-λ +Peptides wereConjugated to Ovalbumin

Example 4 Evaluation of the Ability of Anti-Human IL-21 Antibodies toCross-React and Bind the Human γc-Family Cytokines IL-2, IL-4, IL-7,IL-9, IL-15

Another important characteristic of a specific antibody is the abilityof the antibody to bind to and antagonize the target protein(s) but tonot bind related proteins (non-target) to a significant degree. Theability of anti-human IL-21 antibodies to bind to related cytokines wastested in the Western blot format. Samples of all members of theγc-cytokine family were obtained and run on SDS-PAGE and transferred tonitrocellulose membranes for blotting. Recombinant human IL-2(202-IL/CF), human IL-4 (204-IL/CF), human IL-7 (207-IL/CF), human IL-9(209-IL/CF), human IL-15 (247-IL/CF) all obtained from R&D Systems,Minneapolis Minn.), human IL-21 (ZymoGenetics) and human IFN-λ (U.S.Pat. Nos. 6,927,040; 7,252,969) were used to evaluate the specificity ofthe antibodies. All of the antibodies tested showed no detectablebinding above background to the γc-family cytokines except for humanIL-21 where clear binding was observed, consistent with previous Westernblots using these antibodies (see Example 3).

TABLE 5 Monoclonal Anti-Human IL-21 Antibody Reactivity to γc cytokinesin Western Blot Analysis Hu IFNλ Hu Hu Hu Hu Hu Hu negative Clone IL-2IL-4 IL-7 IL-9 IL-15 IL-21 control 362.597.3 0 0 0 0 0 ++ 0 362.75.1.1 00 0 0 0 +++ 0 362.78.1 0 0 0 0 0 + 0 362.564.1.4 0 0 0 0 0 ++ 0366.328.10.63 0 0 0 0 0 ++ 0 366.552.11.31 0 0 0 0 0 +++ 0 366.617.7 0 00 0 0 +++ 0 Reactivity: None (0) Weak (+) Moderate (++) Strong (+++)

TABLE 6 Monoclonal Mouse and Rat Anti-Human IL-21 Antibody Reactivity inWestern Blot Analysis +Hu IL-21 +Hu IL-21 +Hu IL-21 +Hu IL-21 Hu Cyno MuPeptide Peptide Peptide Peptide Clone* IL-21 IL-21 IL-21 A1744 A1750A1751 A1752 Mouse Clones 338.5.4 0 0 0 0 0 0 0 338.11.5 +++ +++ +/− 0+++ 0 0 338.14.3 + + 0 0 0 0 0 338.15.5 +++ +++ ++ 0 0 0 +++ 338.17.3+++ ++ + 0 0 ++ 0 338.24.5 +++ + 0 0 0 ++ 0 338.25.6 +++ +++ +++ 0 0 0 0338.28.6 +/− 0 0 0 0 0 0 338.29.2 +++ +++ 0 +++ 0 0 0 338.39.5 +++ +++ 00 0 0 0 Rat Clones 272.19.1.1.4.2 +++ +++ +++ ++ 0 0 0 272.21.1.3.4.2+++ +++ + 0 0 0 0 Reactivity: None (0) Weak (+) Moderate (++) Strong(+++) *No Signals Observed with IFN-λ +Peptides were Conjugated toOvalbumin

Example 5 Competitive Epitope Binning Studies

Epitope binning experiments were performed to determine which anti-IL-21monoclonal antibodies are capable of binding simultaneously to humanIL-21. Both human and mouse antibodies were represented. Anti-IL-21monoclonal antibodies that compete for the same, or an overlapping,binding site (epitope) on the antigen are not able to bindsimultaneously and are functionally grouped into a single family or“epitope bin”. Anti-IL-21 monoclonal antibodies that do not compete forthe same binding site on the antigen are able to bind simultaneously andare grouped into separate families or “epitope bins”. Experiments wereperformed using a BIACORE T100™ instrument. Epitope binning experimentswere performed with soluble, (ZymoGenetics) human IL-21 as the antigen.

Epitope binning studies were performed on a BIACORE T100™ system (GEHealthcare, Piscataway, N.J.). Methods were programmed using BIACORET100™ Control Software, v 1.1.1. Individual anti-IL-21 monoclonalantibodies were covalently immobilized to separate flow cells of aBIACORE CM4 sensor chip. Subsequently, the IL-21 antigen was injectedand allowed to specifically bind to the monoclonal antibody immobilizedon the sensor chip. The BIACORE instrument measures the mass of proteinbound to the sensor chip surface, and thus, immobilization of theprimary antibody of a test pair and specific binding of the IL-21antigen to the primary antibody were verified for each test cycle.Following the binding of the IL-21 antigen, a secondary anti-IL-21monoclonal antibody was injected and allowed to bind. If the secondaryanti-IL-21 monoclonal antibody was capable of binding the antigensimultaneously with the primary monoclonal antibody, an increase in masson the surface of the chip, or binding, was detected. If, however, thesecondary anti-Th-21 monoclonal antibody was not capable of binding theantigen simultaneously with the primary monoclonal antibody, noadditional mass, or binding, was detected. Each anti-IL-21 monoclonalantibody tested against itself was used as the negative control toestablish the level of the background (no-binding) signal.

A series of experiments was completed to test the binding properties ofpurified anti-IL-21 monoclonal antibodies obtained from hydridomafusions of the spleens of mice immune to human IL-21. The firstanti-IL-21 monoclonal antibody of a test pair was covalently immobilizedusing EDC:NHS to a density of approximately 1000 RU. The IL-21 antigenwas diluted to 100 nM and allowed to flow over the surface of theimmobilized antibody. Subsequently, the secondary antibody of a testpair was diluted to 5 μg/mL (approximately 32.2 nM) and allowed to bindto the captured IL-21 antigen. A subset of the anti-IL-21 monoclonalantibodies was tested as the primary antibody in combination with thefull panel of secondary anti-IL-21 monoclonal antibodies. Bindingexperiments were performed with a flow rate of 30 μL/min at 25° C. Thebuffer for these studies consisted of 10 mM Hepes, 0.3 M NaCl, 0.05%surfactant P20, 5 mM CaCl₂, 1 mg/mL bovine serum albumin, pH 8.0.Between cycles, the antibody on the chip was regenerated with 20 mMhydrochloric acid. Data was compiled using BIACORE T100™ Evaluationsoftware (version 1.1.1), then loaded into EXCEL™ for additional dataprocessing.

Purified anti-Th-21 monoclonal antibodies were characterized andassigned into epitope bins. The signal (RU, response units) reported bythe BIACORE is directly correlated to the mass on the sensor chipsurface. Once the level of background signal (RU) associated with thenegative controls was established (the same anti-IL-21 monoclonalantibody used as both the primary and secondary antibody), the binningresults were reported as either positive or negative binding. Positivebinding indicates that two different anti-IL-21 monoclonal antibodiesare capable of binding the antigen simultaneously. Negative bindingindicates that two different anti-IL-21 monoclonal antibodies are notcapable of binding the antigen simultaneously. The differential betweenpositive and negative response values in this experiment was significantand allowed for an unambiguous assignment of the anti-IL-21 monoclonalantibodies into six distinct families, or epitope bins. The firstepitope bin was represented by anti-IL-21 monoclonal antibodies from,for example, clones 338.5.4; 362.78.1; and 362.597.3. The second bin wasrepresented by anti-IL-21 monoclonal antibodies from, for example,clones 338.14.3; 362.75.1.1; and 366.328.10. An additional third bin wasfound to overlap the binding epitopes of the bin #1 and bin #2antibodies. It was represented by monoclonal antibody from, for example,clone 366.552.11. Antibodies that neutralize IL-21 are found in each ofthese three bins.

Three additional epitope bins were identified. Each of these bins wasrepresented by monoclonal antibody from, for example, hybridoma clones366.345.6.11 (bin #4), 338.28.6 (bin#5), and 338.39.5 (bin#6). Theantibodies identified in these three bins do not neutralize human IL-21bioactivity.

Example 6 Soluble Receptor Competition Studies

Competition experiments were performed to determine which anti-humanIL-21 monoclonal antibodies are capable of binding IL-21 simultaneouslywith the IL-21 soluble receptor. Anti-human IL-21 monoclonal antibodiesthat compete with the soluble receptor for the same, or an overlapping,binding site (epitope) on the antigen are not able to bindsimultaneously. Anti-IL-21 monoclonal antibodies that do not competewith the soluble receptor for the same binding site on the antigen areable to bind simultaneously. Competition experiments were performed withsoluble, recombinant human IL-21 as the antigen. The IL-21 antigen wasallowed to bind the monoclonal antibody prior to competition with theIL-21 soluble receptor. Two versions of the IL-21 soluble receptor (bothproduced by ZymoGenetics) were utilized for monoclonal antibody analysisin these studies: One version of the receptor consists of a homodimericreceptor (IL-21R-Fc) composed of the extracellular domain of the IL-21receptor fused to an Fc molecule derived from human immunoglobulin. Thesecond soluble receptor form was a heterodimeric receptor (IL-21R/γc-Fc)composed of one subunit comprising the extracellular domain of the IL-21receptor fused to an Fc molecule derived from human immunoglobulin and asecond subunit comprising the extracellular domain of the common γcommon-chain fused to an Fc molecule derived from human immunoglobulin,as described in co-owned U.S. Pat. No. 6,777,539 incorporated byreference herein in its entirety.

Competition studies were performed on a BIACORE T100™ system (GEHealthcare, Piscataway, N.J.). Methods were programmed using BIACORET100™ Control Software, v 1.1.1. Individual anti-IL-21 monoclonalantibodies were covalently immobilized to separate flow cells of aBIACORE CM4 sensor chip. Subsequently, the IL-21 antigen (SEQ ID NO: 2)was injected and allowed to specifically bind to the monoclonal antibodyimmobilized on the sensor chip. The Biacore instrument measures the massof protein bound to the sensor chip surface, and thus, immobilization ofthe primary antibody and specific binding of the IL-21 antigen to theprimary antibody were verified for each test cycle. Following thebinding of the IL-21 antigen, the soluble receptor was injected andallowed to bind. If the soluble receptor was capable of binding theantigen simultaneously with the primary monoclonal antibody, an increasein mass on the surface of the chip, or binding, was detected. If,however, the soluble receptor was not capable of binding the antigensimultaneously with the primary monoclonal antibody, no additional mass,or binding, was detected. Each anti-IL-21 monoclonal antibody testedagainst itself was used as the negative control to establish the levelof the background (no-binding) signal. As a positive control, eachanti-IL-21 monoclonal antibody was tested against an anti-IL-21 antibodyfrom a different epitope bin to determine the level of positive(binding) signal.

A series of experiments were completed to test the binding properties of5 purified anti-IL-21 monoclonal antibodies (from hybridoma clones362.78.1, 366.75.1.1, 366.328.10, 366.552.11.31, and 366.345.6.11) thatbind human IL-21. The first anti-IL-21 monoclonal antibody of a testpair was covalently immobilized using a mixture of 0.4 M EDC[N-ethyl-N′-(3-diethylamino-propyl)carbodiimide] and 0.1 M NHS(N-hydroxysuccinimide) to a density of approximately 1000 RU. Afterimmobilization of the antibody, the active sites on the flow cell wereblocked with 1M ethanolamine. The IL-21 antigen was diluted to 100 nMand allowed to flow over the surface of the immobilized antibody.Subsequently, the soluble receptor was diluted to 10 μg/mL and allowedto bind to the captured IL-21 antigen. Binding experiments wereperformed with a flow rate of 30 μL/min at 25° C. The buffer for thesestudies consisted of 10 mM Hepes, 0.3 M NaCl, 0.05% surfactant P20, 5 mMCaCl₂, 1 mg/mL bovine serum albumin, pH 8.0. Between cycles, the flowcell was washed with 20 mM hydrochloric acid to regenerate the surface.This wash step removed the IL-21 antigen and any bound soluble receptorfrom the immobilized antibody surface, and allowed for the subsequentbinding of the next test sample. Data was compiled using BIACORE T100™Evaluation software (version 1.1.1).

Purified anti-IL-21 monoclonal antibodies were characterized for theirability to compete with the human IL-21 soluble receptor for binding tothe IL-21 antigen. The signal (RU, response units) reported by theBIACORE is directly correlated to the mass on the sensor chip surface.Once the level of background signal (RU) associated with the negativecontrols was established (the same anti-IL-21 monoclonal antibody usedas both the primary and secondary antibody), the competition resultswere reported as either positive or negative binding. Positive bindingindicates that the anti-IL-21 monoclonal antibody and IL-21 solublereceptor are capable of binding the antigen simultaneously. Negativebinding indicates that the anti-IL-21 monoclonal antibody and the IL-21soluble receptor are not capable of binding the antigen simultaneously.The differential between positive and negative response values in thisexperiment was significant and allowed for an unambiguous determinationof competition between the anti-IL-21 monoclonal antibodies and IL-21soluble receptor.

Monoclonal antibodies from hybridoma clones 362.78.1 and 366.552.11.31competed with both versions of the IL-21 soluble receptor (homodimericIL-21R-Fc and heterodimeric IL-21R/γc-Fc) for binding to the human IL-21antigen. Monoclonal antibodies from hybridoma clones 366.328.10 and366.345.6.11 did not compete with either version of the soluble receptorfor binding to the antigen. The monoclonal antibody from hybridoma clone362.75.1.1 showed partial competition for binding with both forms of thesoluble receptor. These studies were performed with the IL-21 antigenpre-bound to the monoclonal antibody. Three of these antibodies(362.78.1, 366.328.10, 366.552.11.31) have been shown to neutralizehuman IL-21 while the monoclonal antibody antibodies from hybridomaclones 362.75.1.1 and 366.345.6.11 are, depending on the assay, veryweak, or non-neutralizers of human IL-21 bioactivity.

Example 7 IL-21 Baf3/huIL-21R STAT3 Bioactivity Assay

The following phosphorylated-STAT3 bioassay was used as a primary screento measure neutralizing anti-IL-21 titers in murine serum as well asrelative levels of IL-21 neutralization by hybridoma supernatants andpurified anti-IL-21 antibodies. IL-21 activity was determined bymeasuring the level of STAT3-phosphorylation following ligand-receptorinteraction in Baf3/KZ134/huIL-21R cells (see Spolski and Leonard, AnnuRev Immunol. Nov. 8, 2007). Relative neutralization activity wasdetermined based on the decrease in phosphorylated-STAT3 levels using anEC₅₀ concentration of IL-21 and a titration of antagonist.

Baf3/KZ134/huIL-21R cells were washed two times with assay media (RPMI1640 with 5% fetal bovine serum, 1× Glutamax, 1% Sodium Pyruvate, and 2μM β-Mercaptoethanol; all from Invitrogen, Carlsbad, Calif.) beforebeing plated out at 40,000 cells/well in 96-well, round-bottom tissueculture plates (Becton Dickinson, Franklin Lakes, N.J.). Cells wereplaced in a 37° C. tissue culture incubator while the test solutionswere prepared. To determine EC₅₀ and EC₉₀ concentrations of IL-21 inthis assay, serial dilutions of recombinant human IL-21 were prepared inassay media and plated in a separate 96-well U-bottom plate.Alternatively, to test for IL-21 neutralization, an EC₅₀ concentrationof IL-21 (determined to be 33 pM) was preincubated with serial dilutionsof IL-21-immunized mouse serum, spent hybridoma media, purified solublehuman IL-21R/γc-Fc or purified monoclonal anti-IL-21 antibodies. Boththe cell plate and the solution plate were then incubated in ahumidified tissue culture chamber to equilibrate for 30 minutes at 37°C. and 5% CO₂. After 30 minutes, the reaction was initiated bytransferring the IL-21 solutions to the cell plate and incubating for 10minutes at 37° C. and 5% CO₂.

Following the 10 minute incubation, reactions were stopped by placingthe plate on ice and adding 125 μL of ice-cold Cell Wash Buffer(BIO-PLEX Cell Lysis Kit, BIO-RAD Laboratories, Hercules, Calif.) toeach well. Cells were then spun down at 1500 rpm at 4° C. for 5 minutesand the media aspirated. To lyse the cells, 50 μL/well Lysis Buffer(prepared according to the manufacturer's instructions, BIO-RAD Labs)was added to each well. The cell lysates were then pipetted up and downfive times while on ice, and agitated on a microplate platform shakerfor 20 minutes at 600 rpm at 4° C. Plates were then centrifuged at 3000rpm at 4° C. for 20 minutes. Supernatants were collected and transferredto a new micro titer plate and mixed 1:1 with Assay Buffer (BIO-RAD) forstorage at −20° C.

Capture beads (BIO-PLEX Phospho-STAT3 Assay, BIO-RAD Laboratories) werediluted and plated in a 96-well filter plate (Millipore Corporation,Ireland) according to manufacturer's instructions. Plates were washedtwo times with Wash Buffer (BIO-RAD) and 50 μL of cell lysate mix wastransferred to each well. Each plate was then wrapped in aluminum foiland shaken overnight at room temperature and 300 rpm. The following day,the plate was transferred to a microtiter vacuum apparatus and washedtwo times with Wash Buffer. After addition of 25 μL/well detectionantibody (BIO-RAD), the foil-covered plate was incubated at roomtemperature for 30 minutes with shaking at 300 rpm. The plate wasfiltered and washed two times with wash buffer. Streptavidin-PE(BIO-RAD; 50 μL/well) was added, and the foil-covered plate wasincubated at room temperature for 15 minutes with shaking at 300 rpm.The plate was filtered and washed two times and resuspended in 125μL/well Bead Resuspension Buffer (BIO-RAD). The level ofphosphorylated-STAT3 was then assessed using an array reader (BIO-PLEX,BIO-RAD Laboratories) according to the manufacturer's instructions. Datawere analyzed using analytical software (BIO-PLEX MANAGER 3.0, BIO-RADLaboratories). Increases in the level of the phosphorylated STAT3transcription factor present in the lysates were indicative of an IL-21receptor-ligand interaction. For the neutralization assay, decreases inthe level of the phosphorylated STAT3 transcription factor present inthe lysates were indicative of neutralization of the IL-21receptor-ligand interaction. IC₅₀ (concentration of antagonist thatyields 50 percent inhibition of ligand activity) values were calculatedusing GraphPad Prism®4 software (GraphPad Software, Inc., San DiegoCalif.) and expressed as molar concentrations for each reagent in theneutralization assay.

Human IL-21 induced STAT3 phosphorylation in a dose dependent mannerwith an EC₅₀ concentration determined to be approximately 33 pM. Table 7summarizes the IC₅₀ values for the positive control (soluble humanIL-21R/γc-Fc fusion protein) and the IL-21 neutralizing entitiesdescribed herein. These data indicate that the IL-21 neutralizingantibodies were active and were equal to or better than the positivecontrol at reducing IL-21-induced STAT3 phosphorylation.

TABLE 7 IC₅₀ Values in STAT3-Phosphorylation Assay IC₅₀ (pM) IC₅₀ (pM)IC₅₀ (pM) Expt #1 Expt #2 Expt #3* soluble hIL-21R/γc-Fc 25 102 140.2IL-21 mAb Clone # 362.75.1 No Neut. 362.78.1 14 60 362.78.1.44 41 66.7362.78-CHO (A2162F) 42.0 366.328.10.63 210 366.552.11.31 59 366.617.7 69366.345.6.11 Weak *Expt 3 conducted using 96 pM IL-21

Example 8 IL-21 Baf3/huIL-21R STAT-Luciferase Bioactivity Assay

This 24-hour assay measures IL-21-induced STAT-Luciferase activity inBaf3/KZ134/huIL-21R transfected cells. Baf3/KZ134/huIL-21R transfectedcells were washed two times with assay media (phenol red-free RPMI 1640with 5% fetal bovine serum, 1× Glutamax, 1% Sodium Pyruvate, and 2 μMβ-Mercaptoethanol; all from Invitrogen, Carlsbad, Calif.) before beingplated out at 40,000 cells/well in a 96-well, flat-bottom opaque whiteculture plates (Corning/Costar, Lowell, Mass.). Cells were then placedin a tissue culture incubator while the test solutions were prepared. Ina separate plate, human IL-21 was mixed with either media or a range ofIL-21 antagonists (either monoclonal antibodies or the soluble humanIL-21 receptor/γc-Fc). Once mixed, this plate was also transferred to ahumidified 37° C. tissue culture incubator. After 30 minutes the testsolutions were transferred to the cell plate and mixed. This plate wasthen placed back in the incubator for 24 hours. After 24 hours, thecells were removed from the incubator and allowed to cool to roomtemperature. Each well was then diluted 1:1 with a 100 μL volume ofSteady-Glo Luciferase reagent (Promega, Madison, Wis.) and mixedthoroughly. The plate was covered and shaken at room temperature for 10minutes and Relative Luciferase Units (RLU) were measured on aluminometer.

To determine EC₅₀ and EC₉₀ concentrations of IL-21 in this assay, serialdilutions of recombinant human IL-21 ranging from 0 to 100 ng/mL weretested. The EC₉₀ concentration of IL-21, ˜15 ng/mL (961 pM), was used insubsequent neutralization experiments. In these experiments, clones362.78.1 (and its subclone 362.78.1.44 and CHO-expressed counterpart,362.78-CHO; see Example 1) and 362.328.10.63 demonstrated the mostpotent anti-IL-21 activity, with IC₅₀ concentrations in the range of300-850 pM, while the IC₅₀ values for the soluble human IL-21receptor/γc-Fc control ranged from 650-1830 pM. The relative activitiesof the neutralizing entities described herein are summarized in Table 8.

TABLE 8 IC₅₀ Values in 24 hr STAT-Luciferase Assay IC₅₀ (pM) IC₅₀ (pM)IC₅₀ (pM) Expt #1 Expt #2 Expt #3 Soluble hIL-21R/γc-Fc 650-850 1830IL-21 mAb Clone# 362.75.1 No Neut. 362.78.1  400 775 760 362.78.1.44 850362.78CHO (A2162F) 533 366.328.10.63 300-500 366.552.11 2400 366.617.76360 366.345.6.11 3184

Example 9 Cross-Reaction to Cynomolgus Monkey, Murine or Rat IL-21Activity

Species cross-reaction studies (especially for non-human primatecross-reactivity) are important to complete prior to pre-clinicalpharmacology/toxicology studies when developing a therapeuticantagonist. In order to determine whether the anti-human IL-21neutralizing entities described herein might cross-react and neutralizethe activity induced by cynomolgus IL-21, murine IL-21 or rat IL-21 (andtherefore, justify either cynomolgus monkeys, mice and/or rats as viabletest species), it was first necessary to demonstrate recombinantcynomolgus, murine and rat IL-21 bioactivity. The methods for IL-21STAT-Luciferase activity assays described in Example 8 were used todetermine EC₅₀ and EC₉₀ values for recombinant human IL-21, cynomolgusIL-21, murine IL-21, and rat IL-21 (all produced at ZymoGenetics).Results indicate that the levels of STAT-Luciferase activity induced bythe human, cynomolgus and murine IL-21 in this assay differ widely. EC₉₀values used in subsequent experiments were determined to be as follows:961 pM for human IL-21; 102 pM for cynomolgus monkey IL-21; 6.41 nM formouse IL-21, and 1.08 nM for rat IL-21. IL-21 soluble receptor(hIL-21R/γc-Fc) neutralized the effects of cynomolgus, murine and rat.

IL-21. Addition of the purified anti-IL-21 monoclonal antibodies shownin Table 9 neutralized cynomolgus IL-21 to varying degrees but did notneutralize murine or rat IL-21 (note that only 362.78-CHO and 366.552.11were tested against rat IL-21). The IC₅₀ values for neutralization ofcynomolgus IL-21 by the neutralizing entities described herein rangedfrom ˜100 pM to 431 pM and are summarized in Table 9. It should be notedthat the best human IL-21 neutralizing antibodies were all able toeffectively neutralize cynomolgus IL-21 but not murine IL-21 or ratIL-21. Additionally, the CHO-cell produced IL-21 mAb (362.78-CHO) wastested in a separate experiment using an 800 pM concentration ofcynomolgus monkey IL-21.

TABLE 9 Cross-reaction of hIL-21 antagonists to cynomolgus monkey,murine and rat IL-21 in the STAT-Luciferase assay. Cyno IL-21 Murine RatIL-21 IC₅₀ IL-21 IC50 IC₅₀ ([cIL-21] = ([mIL-21] = ([rIL-21] = 100 pM)6.41 nM) 1.08 nM) Molar Molar Molar ng/mL conc. ng/mL conc. ng/mL conc.soluble hIL- 20 385 pM 425.8 8.06 707.5 13.4 21R/γc-Fc nM nM IL-21 mAbClone: 362.75.1 weak — none none N/T N/T 362.78.1 60 400 pM none noneN/T N/T 362.78-CHO 255   1.7 nM none none none none (A2162F)* 366.328.1015 100 pM none none N/T N/T 366.552.11 23 156 pM none none none none366.617.7 65 431 pM none none N/T N/T *Note: Clone 362.78-CHO (Example1b) was tested using an 800 pM concentration of cIL-21 instead of 100 pMas was used in other experiments. N/T = not tested.

Example 10 Evaluation of Potential Cross-Reaction to IL-4 in aCell-Based Assay

When developing a therapeutic cytokine antagonist, it is important toknow if it will cross-react with and neutralize structurally relatedcytokines. This primary B cell proliferation assay was designed to testthe IL-21 neutralizing entities described herein for cross-reaction toand neutralization of human IL-4.

Isolation of primary B cells: To obtain primary B cells, 200 mLperipheral blood was collected from healthy human volunteers(ZymoGenetics). Blood was diluted to 400 ml with room temperature PBSand 35 mL aliquots were made in 50 ml conical tubes. Fourteen mL of roomtemperature Ficoll/Hypaque (Pharmacia, Uppsala, Sweden) was underlaidand the tubes were spun for 20 minutes at 2000 rpm. The PBMC interfacelayer was aspirated and washed two times with MACS buffer (PBS, HEPES 20mM, and 1% BSA; Invitrogen, Carlsbad, Calif.). Cells were counted and Bcells were negatively selected using the B Cell Isolation Kit fromMiltenyi Biotec (Auburn, Calif.) following the protocol outlined by themanufacturer. A small sample of the purified B cells were tested forpurity by FACS analysis and found to be >97% pure CD19⁺ B cells in allexperiments.

Proliferation assay: B cells proliferate in response to IL-4 co-culturewith immobilized anti-IgM. To determine potential cross-reaction to andneutralization of IL-4 by anti-IL-21 mAbs, previously isolated B cellswere first plated at 40,000-50,000 cells/well in a 96-well U-bottomtissue culture plate (Becton Dickinson, Franklin Lakes, N.J.) that hadbeen pre-coated with 1.0 μg/mL anti-IgM (Southern Biotech, Birmingham,Ala.). The cells were then treated with 10 ng/mL recombinant human IL-4(R&D Systems; Minneapolis, Minn.) and a titration series of an IL-21antagonist (test antibodies or controls). The cells were then incubatedfor 3 days at 37° C. and 5% CO₂ in a humidified tissue cultureincubator. After three days, the cells were pulsed with 1 μCi/well of[³H]-Thymidine (Amersham Biosciences, Piscataway, N.J.). After 16 hours,the cells were harvested onto glass-fiber filters and the amount of[³H]-incorporation was quantitated using a beta counter (Topcount NXT,Packard). None of the three anti-IL-21 monoclonal antibodies tested(362.78.1, 366.328.10.6 and 366.552.11.31) showed any neutralization ofIL-4-induced proliferation at up to a 250-fold molar excess.

Example 11

11A. Evaluation of Potential Cross-Reaction to IL-2 and IL-15 in aCell-Based Assay

When developing a therapeutic cytokine antagonist, it is important todetermine if it will cross-react with and neutralize structurallyrelated cytokines. The murine T cell line, CTLL-2, can be induced toproliferate in response to human IL-2 or IL-15. Therefore, this assaywas chosen to test the IL-21 neutralizing entities described herein forcross-reaction to and neutralization of human IL-2 and IL-15.

CTLL-2 cells were washed three times in proliferation bioassay media(RPMI 1640, 2× Glutamax, 10% FBS, 2×NaPyr, 1×B-mercaptoethanol and 20 mMHepes; Invitrogen, Carlsbad, Calif.), and plated at 50000 cells per wellin 96-well round bottom tissue culture plates (Becton Dickinson,Franklin Falls, N.J.). To these cells, a predetermined EC₉₀ dose ofeither IL-2 (3.0 ng/mL) or IL-15 (0.5 ng/mL) in combination with aserial dilution of the IL-21 neutralizing entities was added. The ratioof cytokine to antibody ranged from a 250-fold molar excess to a 1:1ratio. Anti-IL-2 or anti-IL-15 neutralizing antibodies (both from R&DSystems, Minneapolis, Minn.) were used as positive controls. The cellswere then incubated for 24 hours at 37° C. and 5% CO₂ in a humidifiedtissue culture incubator. After 24 hours, the cells were pulsed with 1μCi/well of [³H]-Thymidine (Amersham Biosciences, Piscataway, N.J.).Sixteen hours later, the cells were harvested onto glass-fiber filtersand the amount of [³H]-incorporation was quantitated using a betacounter (Topcount NXT, Packard).

Results: None of the three anti-IL-21 monoclonal antibodies tested(362.78.1, 366.328.10.6 and 366.552.11.31) showed any neutralization ofIL-2 or IL-15-induced proliferation.

11B.—Confirmation Using Surface Plasmon Resonance (Biacore) that IL-21mAb 362.78-CHO does not Bind IL-21-Related Human Cytokines IL-2, IL-4,IL-7, IL-9 or IL-15.

The anti-IL-21 mAb 362.78-CHO was evaluated via surface plasmonresonance for potential cross reactivity to human IL-2, human IL-4,human IL-7, human IL-9, and human IL-15.

Materials and Methods: Experiments were completed to test the crossreactivity of the anti-IL-21 monoclonal antibody 362.78-CHO for humanIL-2, human IL-4, human IL-7, human IL-9, and human IL-15. Bindingstudies were performed on a BIACORE T100™ (GE Healthcare, Piscataway,N.J.). Methods were programmed using BIACORE T100™ Control Software, v2.0. Goat anti-human IgG Fc-gamma specific antibody (JacksonImmunoResearch, West Grove, Pa.) was covalently immobilized to flowcells 1 and 2 of a CM4 sensor chip using amine coupling chemistry(EDC:NHS). The purified anti-IL-21 monoclonal antibody 362.78-CHO wassubsequently captured onto flow cell 2 of the sensor chip at a densityof approximately 240 RU. Flow cell 1 was used as the reference surface.

IL-2, IL-4, IL-7, IL-9, and IL-15 (all purchased from R&D Systems,Minneapolis, Minn.) were injected over the captured antibody surface(flow cell 2) and the reference flow cell (flow cell 1) atconcentrations of 100, 20, and 4 nM. As a positive control for this setof experiments, IL-21 (produced at ZymoGenetics) was also injected atidentical concentrations. Binding studies were performed with a flowrate of 25 μL/min, an association time of 2 minutes, and a dissociationtime of 3 minutes. All binding experiments were performed at 25° C. in abuffer of 10 mM HEPES, 300 mM NaCl, 5 mM CaCl₂, 0.05% Surfactant P20(Biacore), 1 mg/mL bovine serum albumin, pH 8.0. Between cycles, theflow cell was washed with 20 mM hydrochloric acid to regenerate thesurface. This wash step removed both the captured 362.78-CHO antibodyand any bound antigen from the chip surface. Data was compiled usingBIACORE T100™ Evaluation software (version 2.0). Data was processed bysubtracting reference flow cell and blank injections. Baseline stabilitywas assessed to ensure that the regeneration step provided a consistentbinding surface throughout the sequence of injections.

Results:

No binding of IL-2, IL-4, IL-7, IL-9, or IL-15 to the 362.78-CHOantibody was observed. In contrast, the IL-21 positive controldemonstrated a dose dependent binding that was consistent with previousstudies.

This lack of cross-reactivity was subsequently explained by epitopemapping studies of clone 362.78 (see Example 17). The amino acidslocated in and near the D-helix of IL-21 shown to be bound by clone362.78 (EKKPPKEFLERFKSLL; SEQ ID NO: 2 from residue 129 to 144)) are notwell-conserved among the related human gamma-chain cytokines, nor withinmouse IL-21, as shown below in Table 10.

TABLE 10 IL21_HUMAN ------TCPSCDSYEKK--PPKEFLERFKSLLQKMIHQHLSSTHGSEDSIL21_MOUSE ------KCPSCDSYEKR--TPKEFLERLKWLLQKMIHQHLS IL15_HUMAN-------CKECEELEEK--NIKEFLQSFVHIVQMFINTS IL2_HUMAN------TTFMCEYADET-ATIVEFLNRWITFCQSIISTLT IL4_HUMAN---GLAGLNSCPVKEANQSTLENFLERLKTIMREKYSKCSS IL7_HUMAN----SLEENKSLKEQKK-LNDLCFLKRLLQEIKTCWNKILMGTKEH IL9_HUMAN------CEQPCNQTTAG--NALTFLKSLLEIFQKEKMRGMRGKI

IL-21 human is shown as SEQ ID NO:2; IL-21 mouse is shown as SEQ IDNO:11; IL-15 human is shown as SEQ ID NO:92; IL-2 human is shown as SEQID NO:93; IL-4 human is shown as SEQ ID NO:94; IL-7 human is shown asSEQ ID NO:95; IL-9 human is shown as SEQ ID NO:96.

Example 12 B Cell Proliferation Assays

Primary B Cell Assays

To further test the activity of the IL-21 neutralizing entities, twoprimary B cell assays were developed. The B cell proliferation assay wasused to demonstrate neutralization of IL-21 induced proliferation over 4days and the B cell differentiation assay demonstrated theneutralization of IL-21 induced plasma cell differentiation over 8 days.These experiments demonstrated neutralization of IL-21 in long-termbiologically relevant assays.

Isolation of primary human B cells: To obtain primary B cells, 200 mLperipheral blood was collected from healthy human volunteers(ZymoGenetics). Blood was diluted with 200 mL of room temperature PBSand 35 mL aliquots were made in 50 mL conical tubes. Fourteen mL of roomtemperature Ficoll/Hypaque (Pharmacia, Uppsala, Sweden) was underlaidand the tubes were spun for 20 minutes at 2000 rpm. The PBMC interfacelayer was aspirated and washed two times with MACS buffer (PBS, HEPES 20mM, and 1% BSA; Invitrogen, Carlsbad, Calif.). Cells were counted and Bcells were negatively selected using the B Cell Isolation Kit fromMiltenyi Biotec (Auburn, Calif.) following the protocol outlined by themanufacturer. A small sample of the purified B cells were tested forpurity by FACS analysis and found to be >97% pure in all experiments.

Proliferation assay: B cells proliferate in response to co-culture withanti-CD40 and IL-21. To determine the neutralization activity of theanti-IL-21 mAbs, B cells were plated at 40000-50000 cells/well in a96-well U-bottom tissue culture treated plate (Becton Dickinson,Franklin Lakes, N.J.). The cells were then treated with 0.1 μg/mLanti-CD40 (goat anti-human CD40 polyclonal; R&D Systems, Minneapolis,Minn.), 50 ng/mL (3.21 nM) recombinant IL-21 (ZymoGenetics, A1207F) anda titration of an IL-21 antagonist (test mAbs or controls). The plate ofcells was then incubated for 3 days at 37° C. and 5% CO₂ in a humidifiedincubator. After three days, the cells were pulsed with 1 μCi/well of[³H]-Thymidine (Amersham Biosciences, Piscataway, N.J.). After 16 hours,the cells were then harvested onto glass-fiber filters and the amount of[³H]-incorporation was quantitated using a beta counter (Topcount NXT,Packard). IC₅₀ curves measuring the effective neutralization ofIL-21-induced proliferation were calculated and expressed as a molarconcentration. The IC₅₀ values for the top neutralizing mAbs describedherein ranged from 0.71 nM to 6.55 nM and are summarized in Table 11:

TABLE 11 Neutralization of IL-21 in B Cell Proliferation Assay IL-21Antagonist IC50 (nM) soluble hIL-21R/γc-Fc 3.5 362.75.1 NoNeutralization 362.78.1 1.17 366.328.10 0.71 366.552.11 4.75 366.617.76.55

B Cell Differentiation Assay: The differentiation of naïve B cells intoantibody-producing plasma cells is greatly facilitated in vitro whenIL-21 is combined with anti-CD40 and IL-4 (Ettinger et al., J. Immunol.175:7867-79, 2005; Ettinger et al, J. Immunol. 178:2872-82, 2007; Kuchenet al. J. Immunol. 179:5886-96, 2007). To demonstrate activity in alonger term assay than the two Baf3-based screening assays described inExamples 7 and 8, the neutralizing entities described herein were usedto neutralize IL-21 and inhibit human plasma cell differentiation. Toaccomplish this, primary human B cells were plated at 150,000 cells/wellin a 96-well flat bottom tissue culture treated plate (Becton Dickinson,Franklin Lakes, N.J.). The cells were then treated with 0.1 μg/mLanti-CD40 (goat anti-human CD40 polyclonal; R&D Systems, Minneapolis,Minn.), 10 ng/mL recombinant human IL-4 (R&D Systems) and 25 ng/mL (1.6nM) recombinant human IL-21 (ZymoGenetics). IL-21 antagonists (test mAbsor controls) were then added and the cells incubated at 37° C. and 5%CO₂ for eight days in a humidified incubator. At the end of eight days,conditioned medias were collected (for antibody titers) and cellspelleted for subsequent flow cytometry analysis.

B cell analysis using flow cytometry: Cells were resuspended in humanFACS buffer (HBSS, 20 mM HEPES, 1% BSA (all from Invitrogen) and 2%Human Ab Serum (Gemini Bio-Products, Woodland, Calif.)) for five minutesto block Fc receptors. Cells were then centrifuged (5 minutes at 1200rpm) and aspirated. Stains were prepared by diluting antibodies 1:100 inhuman FACS buffer and dispensing 100 μL per sample. Single stains (toadjust cytomer compensation settings) and a multi-stain mixture wereprepared using the following antibodies: anti-CD138-FITC, anti-IgD-PE,anti-CD38-PE-Cy5.5 and anti-CD19-APC. Plasma cells were defined as large(assessed by forward light scatter) CD19⁺, IgD^(lo), CD38⁺ and CD 138⁺cells. The percentage of plasma cells relative to total B cells was usedto determine effectiveness of the neutralizing entities describedherein.

Results: When IL-21 was combined with IL-4 and anti-CD40, approximately50% of the live, large B cells on day 8 were IgD^(low), CD138⁺ plasmacells. Without IL-21, the proportion of plasma cells was ˜8% of thelarge B cells. The addition of the various IL-21 antagonists to theIL-21-containing cultures decreased the proportion of plasma cells in adose-dependent manner. Clone 362.78.1 was the most effectiveneutralizer, and almost completely neutralized IL-21 activity at the10:1 and 2.5:1 antagonist:ligand ratios. The other antibodies tested,clones 366.328.10.63 and 366.552.11.31 were nearly as effective atneutralizing the IL-21 driven differentiation. This data is summarizedin Table 12.

TABLE 12 Inhibition of human plasma cell differentiation by neutralizinganti-hIL-21 mAbs Antagonist:Ligand Ratio 10:1 2.5:1 0.6:1 0.16:1 IL-21Antagonist % IgD-low, CD138+ Plasma Cells 362.78.1 13.7 15.5 34.1 42.8366.328.10.63 15.4 20.2 41.4 52.1 366.552.11.31* 15.4 26.4 44.8 50.3IL-21 Receptor 23.4 41.7 54.2 66.7 *Note that actual antagonist:ligandratios for clone 366.552.11.31 were 14.4, 3.6, 0.9, and 0.22:1

Example 13 DTH Mouse Model

DTH responses are classic immune responses that are initiated by CD4+ Tcells and mediated by T cells, neutrophils and macrophages. A DTHresponse is a good indicator of a CD4+ T cell mediated response. Miceare immunized sub-cutaneously with chicken ovalbumin protein (OVA) ineither of 2 adjuvants, Complete Freunds Adjuvant (CFA; Sigma) or Ribi(Sigma; aka MPL+TDM+CWS adjuvant). This phase is called thesensitization phase (days 0-6). Ear measurements are taken seven dayslater. Mice are then injected in the ear with control PBS (left ear) orOVA (right ear). This phase is called the challenge phase (days 7-8).Immune responses generated to OVA induce inflammation in the earresulting an increase in ear thickness in 24 hours in the OVA-treated,but not in the PBS-treated ear. This is measured using calipers.

C57BL/6 mice (n=8/group) are immunized in the back with 100 μg chickenovalbumin (OVA) emulsified in CFA in a total volume of 200 μl. If Ribiis used instead of CFA, 0.5 mg/ml of ovalbumin is added to a single vialof RIBI and vortexed vigorously for 2 minutes to form an emulsion thatis used to inject mice. Seven days after the immunization, mice areinjected with 10 μl PBS in the left ear (control) and with 10 μg OVA inPBS in the right ear in a volume of 10 μl. Ear thickness of all mice ismeasured before injecting mice in the ear (0 measurement). Ear thicknessis measured 24 hours after challenge. The difference in ear thicknessbetween the 0 measurement and the 24 hour measurement is calculated andis reflective of the inflammation in the ear. Groups of mice areinjected with PBS or different concentration of anti-IL-21 antibodyintra-peritoneally from either days 0-6 (sensitization phase) or fromdays 7-8 (challenge phase). The injection on day 7 and 8 is given 2hours before measuring ear thickness at the 0 and 24 hour time points.At the end of the 24 hour period, once ear thickness was measured, theears were cut and placed in formalin for histological analysis.

Example 14 Mouse Model for Multiple Sclerosis

To test if anti-IL-21 has any effects on multiple sclerosis, the abilityof anti-IL-21 antibodies to inhibit experimental autoimmuneencephalomyelitis (EAE-MS), a mouse model for MS is tested. The wellcharacterized T cell-dependent myelin oligodendrocyte glycoprotein (MOG)35-55 peptide immunization model in C57BL/6 mice is used. The experimentis run to determine that anti-IL-21 antibody could delay and/or inhibitdisease scores in EAE either by inhibiting DC mediated antigenpresentation or by enhancing CD8 T cell responses. Absence of efficientCD8 T cell responses in this model exacerbates EAE (Malipiero et. al.,Eur. J. Immunol., 27:3151-3160, 1997). Delayed onset of disease in theEAE model in a dose dependent manner suggests that use of anti-IL-21antibody may be beneficial in MS.

EAE is a mouse model for MS. In one such model, C57BL/6 mice areimmunized with 100 μg MOG peptide (MOG35-55) or 100 μg recombinant MOGprotein emulsified in CFA adjuvant. Two milliliters of a 0.5 mg/mlpreparation of the MOG35-55 in PBS is added to a vial of CFA andvortexed vigorously to emulsify the solution or a 1:1 ratio ofrecombinant MOG in CFA is prepared. The backs of mice are shaved and 100μg MOG/CFA is injected s.c in the backs of mice. Weights of mice aretaken 2 days before and every day after the immunization. Mice are theninjected on day 2 i.v. with 200 μl pertussis toxin (PT), a finalconcentration of 200 ng/mouse. Mice are monitored daily for clinicalscores. Groups of mice are injected i.p. with 200 μl PBS, 100 μg BSA, 10μg-200 μg anti-IL-21 antibody in a 200 μl volume from days 0-20, or 3× aweek for 3 weeks. The weights of mice, clinical scores and incidence areevaluated and plotted for analysis.

Example 15 Anti-mIL-21 Antibody Decreases Disease Incidence andProgression in a Mouse Model of T-Cell Adoptive Transfer Colitis andPsoriasis

Adoptive transfer of naive T cells into minor histocompatibilitymismatched or syngeneic immunocompromised mice leads to development ofcolitis (Leach M W et al 1996, Powrie F et al, 1997) as well as skinlesions resembling psoriasis (Schon M P et al., Nat. Med. 2:183-8, 1997;Davenport C M et al., Int Immunopharmacol. 5:653-72, 2002).Transplantation of as few as 0.2 million CD4+CD25− T cells from BALB/Cor B10.D2 mice into immunocompromised C.B-17 SCID mice results in weightloss, hemoccult positive stool and development of skin lesions. Thesymptoms in these mice vary from colony to colony.

This model of colitis/psoriasis has some similarities to human Crohn'sdisease and psoriasis, and has been used extensively to test efficacy oftherapeutics for these diseases in humans. For this experiment, mice (8B10.D2 female mice donors; 50 C.B-17 SCID female recipients) wereobtained from Jackson Laboratories or Charles River Laboratories,respectively. Spleens from 8 B10.D2 mice were collected. CD4+ CD25−T-cell were isolated from pooled spleens using standard methodologyknown in the art. Purity of the T-cell population was evaluated by flowcytometry.

Naïve C.B-17 SCID mice received 5×10⁵ CD4+ CD25− T-cells (isolated fromspleens of B10.D2 mice) via intravenous injection on day 0. All micewere weighed at least five times per week and carefully observed forweight loss, which can be associated with colitis. In addition, aclinical colitis score [stool consistency and blood in stool] was takenat least one day per week. Mice were also carefully monitored at leastfive days per week and assigned a score for signs of psoriatic symptoms(hair loss, scratching, alopecia, etc).

A rat anti-mouse IL-21 (mIL-21) antibody, a rat isotype controlantibody, or vehicle (PBS) was administered to groups of mice beginningon day 0. The treatments were delivered as intraperitoneal injections,twice a week, with the antibodies being administered at 0.2 or 0.8 mgper mouse per dose. They could also be delivered using a similar dosingregimen or other route of administration. There were 9-10 mice per groupin the anti-IL21 antibody groups, 6-7 mice per group in the isotypecontrol antibody groups, and 10 mice per group in the PBS group. Thisdosing regimen is referred to as “prophylactic dosing”.

In a separate experiment, groups of mice dosed with the same antibodiesand doses described above (10 mice per group) started their treatmentson day 12 following cell transfer, which is approximately the day thatthe mice began showing signs of psoriasis and/or colitis. This dosingregimen is referred to as “therapeutic dosing”.

At the end of the study (day 45), colonic tissue was submitted forhistological evidence of colitis and serum for analysis of cytokine andchemokine levels.

Results of Prophylactic Dosing:

Mice receiving the anti-mIL-21 antibody at both the 0.2 and 0.8 mg doseswere characterized by significant (at least p<0.05 or better) reductionsin body weight loss and significant reductions in psoriatic skin andcolitis symptom throughout the experimental period compared to miceadministered PBS or 0.2 mg isotype control monoclonal antibody. At theend of the study (day 45), mice treated with either dose of the mIL-21antibody were at approximately 100% of their starting body weight,whereas PBS-treated mice had lost an average of 16% of their startingbody weight and mice treated with an isotype control antibody had lost10-15% of their starting body weight. At day 45, mice treated witheither dose of the mIL-21 antibody had approximately 6.5-7-fold loweraverage colitis clinical scores and approximately 5-7 fold lower averagepsoriatic skin scores. Only 20% of mice treated with 0.2 mg anti-mIL-21antibody developed psoriasis, which was mild, whereas none of the micetreated with the 0.8 mg dose developed any psoriatic skin symptoms. Onthe other hand, 100% of the PBS-treated mice developed psoriasis, withapproximately 50% of these mice developing severe symptoms. At the endof the study, there was also a significant reduction in histologicindices of colitis (scored for intestinal inflammation, lesions, andarchitecture) in the anti-mIL-21 antibody treated mice compared to PBS-and 0.2 mg isotype-control treated mice.

Mice treated with anti-mIL-21 antibody had significantly lower serumIL-6, RANTES, TNF-α, and MIP-1β levels compared to PBS-treated mice,further supporting an anti-inflammatory role for anti-mIL-21 antibody.

Results of Therapeutic Dosing: Mice receiving the anti-mIL-21 antibodyat both the 0.2 and 0.8 mg doses, beginning from day 12 following T celltransfers, were characterized by reductions in body weight loss andsignificant reductions in colitis symptoms throughout the experimentalperiod compared to mice administered the isotype control monoclonalantibody. At day 45, mice treated with either dose of the anti-mIL-21antibody had approximately 3.5-4-fold lower average colitis clinicalscores compared to isotype control antibody-treated mice. Mice treatedwith the 0.8 mg dose of anti-mIL-21 antibody had lower psoriasis scoresthan isotype control antibody- or PBS-treated mice.

Summary: Taken together, these results indicate that in vivoadministration of an anti-mIL-21 antibody was efficacious in reducingcolitis and psoriasis onset and severity in a murine T cell transfermodel, and suggest that anti-IL-21 antibodies may be efficacious intreating human inflammatory bowel disease and/or psoriasis.

Example 16 Contact Hypersensitivity Mouse Model

Contact hypersensitivity can be induced in mice using a variety ofcontact allergens including dinitrofluorobenzene (DNFB) and oxazolone.Mice are sensitized topically with the allergen in a vehicle of acetoneand olive oil and then challenged in the ear with the allergen in oliveoil alone. Change in ear thickness is a measure of the immune responseagainst the allergen. Anti-IL-21 antibodies are administered either atthe sensitization phase (days 0-5) or during the challenge phase (days5-6). Inhibition of ear thickness by antagonizing IL-21 indicates a rolefor IL-21 in inducing contact hypersensitivity.

C57Bl/6 mice are painted on the back with 0.5% DNFB in acetone:olive oil(4:1) or acetone:olive oil alone on day 0. On day 5, ear thickness ofmice is measured using calipers and mice are challenged in the ears witholive oil alone (control) or 0.25% DNFB in olive oil by dropping a 25 μlsolution onto the ear. Change in ear thickness is measured on day 6 andthe inflammation calculated as a difference in ear thickness between day5 and day 6. Groups of mice are injected i.p. with PBS or 10-100 μganti-IL-21 antibodies on either days 0-5 or days 5-6.

Inhibition of ear thickness by anti-IL-21 antibodies demonstrates thatanti-IL-21 antibodies can be useful in inhibiting contacthypersensitivity.

Example 17 Epitope Mapping

A. Hydrogen-Deuterium Exchange (HDx) experiment

In an effort to identify the epitope regions of IL-21 recognized byneutralizing anti-IL-21 mAbs 362.78.1.44, 362.597.3, 366.328.10,366.552.11 and the soluble hIL-21R/γc-Fc an immunoaffinity-basedhydrogen deuterium exchange (HDx) method was applied followed by massspectrometry analysis. Specifically, the purified mAbs were immobilizedon CNBr-activated sepharose beads and exchanged into deuterium buffer.Deuterated IL-21 was bound to the immunoaffinity beads by incubation andthe beads were washed with deuterated buffer to remove unbound proteins.The antigen-antibody complex was then subjected to PBS solution toinitiate back-exchange to amide hydrogen on the unbound regions ofIL-21. Deuterium hydrogen exchange was then quenched and IL-21 waseluted in low pH buffer, which was then subjected to proteolyticdigestion by immobilized pepsin. Peptide mass maps were then generatedby MALDI-TOF mass spectrometry and compared with that of the controlsample, generated from the free state of IL-21, which was exchanged backto amide hydrogen from the deuterated IL-21 by dilution with PBSsolution. FIG. 3 shows expanded mass spectra of pepsin-digested peptidesof both the free-state of IL-21 (FIGS. 3A and 3C) and the antibody-boundIL-21 (FIGS. 3B and 3D). As indicated in FIG. 3A, overlapping peptideisotope were assigned to corresponding peptides EKKPPKEF (SEQ ID NO: 2from residue 129 to 136) (m/z, 1002.5619 Da) and LERFKSLL (SEQ ID NO: 2from residue 137 to 144) (m/z, 1005.6091 Da) of the free-state of IL-21with theoretical peptide masses within 10 ppm mass accuracy. A smallamount of residual deuterated peptide was observed around m/z, 1002.5619Da, due to incomplete amide hydrogen exchange.

FIG. 3B is a spectrum of the same mass range of FIG. 3A showing the twooverlapping peptide isotope envelopes having monoisotope ions at 1014.49m/z and 1015.00 m/z. Because two peptide ions were clustered around thesame mass range, it was difficult to assign each peptide identitybetween the two ions. However, tandem mass spectrometry data of thepeptide ions of FIGS. 3A and B showed that they had identical peptidefragmentation patterns (data not shown). Although there was a smallpercentage of non-deuterated peptide detected from the sample obtainedfrom the antigen-antibody complex, the majority of the peptide ionsretained deuterium and shifted to the higher mass region by virtue oflimited solvent accessibility to the antibody/antigen binding regions,indicating that the mAb binding site likely contains the regionEKKPPKEFLERFKSLL (SEQ ID NO: 2 from residue 129 to 144).

Another pepsin-digested peptide from both the free-state of IL-21 andthe antigen-antibody complex was observed as shown in FIGS. 3C and D andit was identified as KSLLQKMIHQHLSSRTHGSEDS (SEQ ID NO: 2 from residue141 to 162) (m/z, 2519.2451) based on the theoretical mass ofpepsin-digested peptide fragment of IL-21. As shown in FIG. 3D,comparing the mass shift of this 22-amino acid residue peptide(Δmass=9.0 Da) with the mass shift of those two upstream residues (FIG.2B, EKKPPKEF (SEQ ID NO: 2 from residue 129 to 136) and LERFKSLL (SEQ IDNO: 2 from residue 137-144), this region was only marginally protectedupon binding of the mAb, indicating that only a portion of this peptidemay be involved in binding to the mAb. In fact this peptide sequence isthe C-terminal tail and it contains four overlapping amino acid residueswith the peptide (LERFKSLL (SEQ ID NO: 2 from residue 137 to 144), whichappeared to be significantly protected by the mAb. Based on these massspectrometric measurements of deuterium retention, we estimated that theIL-21 epitope for binding of the mAb EKKPPKEFLERFKSLL (SEQ ID NO: 2 fromresidue 129 to 144) and the upstream sequence of KSLLQKMIHQHLSSRTHGSEDS(SEQ ID NO: 2 from residue 141 to 162).

B. Lysine Labeling Protection Experiment

Using the HDx assay, the IL-21 mAb binding epitope region was estimatedand it was observed that five lysine residues (from a total of 12 lysineresidues in IL-21) reside in the estimated binding epitope region. Sincelysine residues are most likely to be present at solvent accessibleregions of proteins due to their charged characteristic, lysine appearsto be an ideal choice for selective chemical modification for a paralleldetermination of the antigen epitope region. The concept behind thechemical modification strategy is that the protection of lysinemodification in an antigen in the presence and absence of antibodycorrelates to its binding epitope (Scholten et al., J. Amer. Soc. MassSpectr. 17: 983-994, 2006). Therefore, for further characterization ofthe epitope region and for the determination of those lysine residuesinvolved in the binding of the mAb, selective acetylation on lysineresidues was performed in both the affinity-bound and free states ofIL-21. The site of lysine modification/protection was determined bywhole mass and peptide mapping analyses using MALDI-TOF and electrosprayionization (ESI) mass spectrometry.

Scholten et al., (Scholten et al., J. Amer. Soc. Mass Spectr. 17:983-994, 2006) investigated the molar ratio between the acetylationreagent (NHS-acetate) and an antigen for the full acetylation of solventaccessible lysine residues and found that the labeling was effective at250-fold molar excess of the reagent in 3 min reaction. To determine thebinding epitope, the same labeling reaction condition was employed forboth the free state IL-21 and the affinity bound IL-21 with severalneutralizing IL-21 mAbs, as well as the IL-21 heterodimeric receptorprotein (IL-21R/γc-Fc). With the given labeling reaction condition,different solvent accessibility of lysine residues of the IL-21 aloneand the affinity-bound IL-21 gave rise to the distribution of lysineacetylation (acetyl occupancy) on the IL-21 molecule. The number ofprotected lysine residues on the affinity-bound IL-21 was compared tothe IL-21 alone by the most intense ion. Spectral alignment based on themost intense ions clearly showed that the number of lysine acetylationson the antigens isolated from different immune complexes is varied. Itwas evident that the lysine labeling reagent was less accessible intothe affinity-bound IL-21. Hence, the acetylation was reduced by thebinding of the antigen to the antibody.

The acetylation protected lysines, which may be involved in the bindingof the mAb, were further probed by protease digestion, and followed bypeptide mass mapping analysis using liquid chromatography massspectrometry. Since covalently modified lysine residues are resistant totryptic digestion, pepsin proteolytic enzyme was used to generate moremass spectrometry-detectable peptides to study the lysine modificationin more detail. The modification of individual peptides using singlepeptide ion chromatography was investigated. As shown in FIG. 4,selected ion chromatograms were generated from both the control (IL-21alone) and the test samples (affinity bound IL-21 molecules) todetermine acetylated and non-acetylated lysine residues. FIG. 4A is aselected ion chromatogram of a proteolytic peptide eluting at 56.22 minin the given chromatographic condition and the monoisotope peptide ionmass was at 662.9 Da, which appeared to be in a triply charged state(Δm=0.3 Da) as indicated in the embedded mass spectrum. Identificationof this peptide in a triply charged state as the lysine acetylatedpepsin-digested peptide fragment, TCPSCDSYEKKPPKEF (SEQ ID NO: 2 fromresidue 119 to 136) (m/z, 1986 Da) was made, whereas the non-acetylatedpeptide mass is 1860 Da (m/z). The mass difference (acetylatedpeptide/non-acetylated peptide) was 126 Da, indicating that all threelysine residues in this peptide were acetylated in the free-state ofIL-21. However, a selected ion chromatogram of the affinity bound IL-21showed no trace of the peptide (FIG. 4B) indicating that the threelysine residues were completely protected by the IL-21 antibody binding.

An additional pepsin-digested peptide (KSLLQKMI (SEQ ID NO: 2 fromresidue 141 to 148) was found to be protected upon binding of IL-21 mAb(FIGS. 4C and 4D). Its monoisotopic ion was at 509 Da (m/z) as a doublycharged ion (Δm=0.5 Da) and the mass difference (acetylatedpeptide/non-acetylated peptide) was 42 Da, indicating that only one ofthe two lysine residues in this peptide was protected. Earlier H/Dexchange experiments indicated that the lysine upstream of theC-terminal tail of the peptide sequence, KSLLQKMIHQHLSSRTHGSEDS (SEQ IDNO: 2 from residue 141 to 162), was most likely protected by theantibody binding. Thus it is most likely that K113 instead of K119 ofthe mature IL-21 molecule (i.e., K141 instead of K147 of SEQ ID NO: 2)is involved in the antibody binding.

Four lysine residues (K102, K103, K106 and K113 of the mature IL-21molecule (i.e., K130, K131, K134, and K141 of SEQ ID NO:2)) wereprotected from acetylation by clones 362.78.1.44 and 362.597.3 bindingand they were located within the estimated IL-21 mAb binding epitoperegion as determined from the HDx assay. Collectively, the acetylationprotection assay provided the involvement of specific lysine residues inthe antigen-antibody interaction and further confirmed the epitopesequence estimated from the HDx assay.

17C.—Comparison of IL-21 Amino Acid Sequences from Various Species

To better understand the species cross-reactivity results in Examples 3and 9, in light of the defined epitopes on IL-21 bound by the IL-21 mAbs(Example 17A and B), amino acid sequences were obtained and compared forIL-21 across multiple species (Table 13). The overall human sequence wasmore than 96% identical to cynomolgus and rhesus monkey sequences, whileonly 61-65% identical to the rodent IL-21 sequences. Notably, thediscontinuous epitope bound by clones 362.78.1.44 and 362.597.3 asdescribed in Example 17 (underlined in Table 13) is identical in human,cynomolgus and rhesus monkey IL-21, while the rat IL-21 sequence differsfrom human IL-21 in 6 residues in these regions, and the mouse IL-21sequence differs by 7 residues.

TABLE 13 IL-21 amino acid sequence alignment for human,cynomolgus monkey, rhesus monkey, rat and mouse IL-21. Hu IL-21MRSSPGNMERIVICLMVIFLGTLVHKSSS QGQDRHMIRMRQLIDIVDQLKNYVNDLV CynoIL-21MRSSPGNMERIVICLMVIFLGTLVHKSSS QGQDRHMIRMRQLIDIVDQLKNYVNDLD RhesusIL-21MRSSPGNMERIVICLMVIFLGTLVHKSSS QGQDRHMIRMRQLIDIVDQLKNYVNDLD Rat IL-21       MERTLVCLILIFLGTVAHKSSP QRPDHLLIRLRHLMDIVEQLKIYENDLD MuIL-21       MERTLVCLVVIFLGTVAHKSSP QGPDRLLIRLRHLIDIVEQLKIYENDLD HuPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPP CynoPEFLPAPEDVETNCEWSAISCFQKAQLKSANTGNNERIINLSIKKLKRKSP RhPEFLPAPEDVETNCEWSAISCFQKAQLKSANTGNNERIINLSIKKLKRKSP RatPELLTAPQDVKGQCEHEAFACFQKAKLKPSNTGNNKTFINDLLAQLRRRLP MuPELLSAPQDVKGHCEHAAFACFQKAKLKPSNPGNNKTFIIDLVAQLRRRLP HuSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS CynoSTGAERRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS RhSTGAERRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS RatAKRTGNKQRHMAKCPSCDLYEKKTPKEFLERLKWLLQKMIHQHLS MuARRGGKKQKHIAKCPSCDSYEKRTPKEFLERLKWLLQKMIHQHLS

IL-21 human is shown as SEQ ID NO:2; IL-21; mouse is shown as SEQ IDNO:11; IL-21 cynomylous is shown as SEQ ID NO:9; IL-21 rhesus is shownas SEQ ID NO:9; IL-21 rat is shown as SEQ ID NO:97.

The discontinuous epitope determined for two highly related anti-hIL-21mAbs 362.78.1 and 362.597.3 is underlined above.

Example 18 Anti-Idiotype Monoclonal Antibodies to 362.78-CHO for Use inPre-Clinical and Clinical Immunoassays

Anti-idiotype mAbs were generated specific for 362.78-CHO forapplication in pre-clinical and clinical immunoassays, such thatpotential anti-362.78-CHO antibody responses in individuals treated withthis therapeutic anti-IL-21 mAb can be specifically measured.

To distinguish between the immunogen (362.78-CHO), which is itself anantibody, and the anti-idiotype antibodies in this Example, theimmunogen will be designated as Ab1 and an anti-idiotype antibody willbe designated as Ab2. An anti-idiotypic antibody should inhibit(neutralize) binding of the Ab1 to its antigen (IL-21). However, itshould be noted that in the process, anti-Ab 1 antibodies will begenerated that are not anti-idiotypic. By definition, these will beanti-362.78-CHO binding, non-neutralizing antibodies and may also be ofuse in the pre-clinical and clinical immunoassays.

Methods: To immunize mice with 362.78-CHO, five 6 to 8 week old BALB/cmice (Charles River Laboratories, Wilmington, Mass.) were immunized with362.78-CHO. The mice were initially immunized by subcutaneous injectionwith ˜50 μg of purified, 362.78-CHO (Lot#A2125F) in combination withEmulsigen®-P adjuvant (MVP Laboratories INC, Omaha, Nebr.) as per themanufacturer's instructions. Following the initial immunization, each ofthe mice received an additional 50 μg of 362.78-CHO in Emulsigen®-Padjuvant via the subcutaneous route every two weeks over a six weekperiod. Seven days after the third and fourth immunizations the micewere bled via the retro orbital plexus and the serum was separated fromthe blood for analysis of its ability to bind to 362.78-CHO.

Selection of fusion animal using both a capture assay and aneutralization assay:

Capture Assay: The ability of mouse anti-362.78-CHO (Ab2, anti-idiotype)antibodies in the antisera to bind to 362.78-CHO (Ab1, produced in CHOcells, lot #E10569) was assessed using a capture style ELISA assay. Inthis assay, wells of 96-well polystyrene ELISA plates were first coatedwith 100 μL/well of goat anti-human IgG, Fc specific antibody (JacksonImmunoResearch Laboratories, West Grove, Pa.) at a concentration of 1μg/mL in Coating Buffer (0.1M Na₂CO₃, pH 9.6). Plates were incubatedovernight at 4° C. after which unbound antibody was aspirated and theplates washed twice with 300 μL/well of Wash Buffer (PBS-Tween definedas 0.137M NaCl, 0.0022M KCl, 0.0067M Na₂HPO₄, 0.0020M KH₂PO₄, 0.05% v/wpolysorbate 20, pH 7.2). Wells were blocked with 200 μL/well of BlockingBuffer (PBS-Tween plus 1% w/v bovine serum albumin (BSA) for 60 minutesat mom temperature (RT), aspirated and the plates washed twice with 300μL/well of PBS-Tween. Wells were incubated with 362.78-CHO (Ab1, ZGIproduced in CHO cells, lot #E10569) at a concentration of 1 μg/ml (in 1%BSA in PBS-Tween). After 1 hour incubation at RT, wells were aspiratedand the plates washed twice as described above. Serial 10-fold dilutions(in 1% BSA in PBS-Tween) of the antisera (Ab2) were prepared beginningwith an initial dilution of 1:1000 and ranging to 1:1,000,000. Duplicatesamples of each dilution were then transferred to the assay plate, 100μL/well. Normal mouse sera served as a negative control. Following a 1hour incubation at RT, the wells were aspirated and the plates washedtwice as described above. Goat anti-mouse IgG, Fc specific, HRPconjugated antibody (Jackson ImmunoResearch Laboratories) at a dilutionof 1:5000 was then added to the wells, 100 μL/well. Following a 1 hourincubation at RT, unbound detection antibody was aspirated from thewells and the plates washed twice. After the aspiration, 100 μL/well oftetramethyl benzidine (TMB) (BioFX Laboratories, Owings Mills, Md.) wasadded to each well and the plates incubated for 1 minutes at RT. Colordevelopment was stopped by the addition of 100 μL/well of Stop Reagent(BioFX Laboratories, Owings Mills, Md.) and the absorbance values of thewells read on a Molecular Devices Spectra MAX 340 instrument at 450 nm.

Neutralization Assay: The ability of mouse anti-362.78-CHO anti-idiotypeantibodies (Ab2) in the antisera to inhibit (neutralize) the bindingactivity of 362.78-CHO (Ab1) was assessed using a plate basedneutralization assay. In this assay, wells of 96-well polystyrene ELISAplates were first coated with 100 μL/well of human IL-21 ligand (lot#A1207F) at a concentration of 1 μg/mL in Coating Buffer (0.1M Na₂CO₃,pH 9.6). Plates were incubated overnight at 4° C., after which unboundligand was aspirated and the plates washed twice with 300 μL/well ofWash Buffer (PBS-Tween defined as 0.137M NaCl, 0.0022M KCl, 0.0067MNa₂HPO₄, 0.0020M KH₂PO₄, 0.05% v/w polysorbate 20, pH 7.2). Wells wereblocked with 200 μL/well of Blocking Buffer (PBS-Tween plus 1% w/vbovine serum albumin (BSA)) for 1 hour, after which the plates werewashed twice with Wash Buffer. Serial 10-fold dilutions (in 1% BSA inPBS-Tween) of the antisera (Ab2) were prepared beginning with an initialdilution of 1:100 and ranging to 1:100,000. Normal mouse sera served asa negative control. Duplicate samples of each dilution were thentransferred to a 96-well dilution plate, 100 μL/well. Ab1 was added as a2× solution, 100 μL/well. Following a 45 minute incubation at RT, 100μL/well was transferred to the assay plate after the Blocking Buffer wasaspirated. Following a 1 hour incubation at RT, the wells were aspiratedand the plates washed twice as described above. Horseradishperoxidase-labeled Goat anti Human IgG, Fc specific, HRP conjugated(Jackson ImmunoResearch Laboratories, West Grove, Pa.) at a dilution of1:5000 was then added to each well, 100 μL/well, and the platesincubated at RT for 1 hour. After removal of unbound detection antibody,the plates were washed twice, 100 μL/well of tetra methyl benzidine(TMB) (BioFX Laboratories, Owings Mills, Md.) added to each well and theplates incubated for 2 minutes at RT. Color development was stopped bythe addition of 100 μL/well of Stop Reagent (BioFX Laboratories, OwingsMills, Md.) and the absorbance values of the wells read on a MolecularDevices Spectra MAX 340 instrument at 450 nm.

Fusion: Two mice with the highest anti-362.78-CHO neutralization titerswere immunized a final time with approximately 50 μg of 362.78-CHO (Ab1)in PBS without adjuvant via subcutaneous injection. Four days later, thespleen and lymph nodes of these mice were harvested. Electrofusion wasperformed using standard methods known in the art to fuse lymphocyteswith mouse myeloma P3-X63-Ag8.653 cells (American Type CultureCollection, CRL 1580) at a 1:1 lymphocyte-myeloma ratio, using theCyto-pulse CEEF-50 apparatus (Cyto Pulse Sciences Inc., Glen Burnie,Md.). The fusion mixture was distributed into 96-well flat-bottomedplates. Wells of the fusion plates were fed three times with a 70%replacement of hybridoma growth medium (IMDM with 1×L-glutamine (100×),1× Penicillin-Streptomycin (100×), all from Gibco Invitrogen, Carlsbad,Calif., 10% Fetalclone1 serum, non-heat inactivated (HyClone, Logan,Utah), 10% Hybridoma Cloning Factor (BM Condimed H1 Roche Diagnostic,Indianapolis, Ind.), 1×HAT supplement (50×, Gibco Invitrogen). Wellswere assayed ten days after plating of the fusion.

Selection of Master Wells: The 96-well fusion plates were screened forthe presence of mouse anti-362.78-CHO idiotype antibodies using acapture style ELISA as described above except that hybridomasupernatants were tested undiluted from the culture plates. Hybridomacells of positive wells were successfully expanded into culture in24-well plates. When the density of the 24-well cultures wasapproximately 4-6×10⁵ cells/mL, the supernatant (approximately 1.5 mL)was individually collected and stored for each well and the cells fromeach well cryopreserved. Freezing medium consisted of 90% Fetalclone1serum and 10% DMSO. Each of the 24-well supernatants was reanalyzed inboth the capture ELISA and plate based neutralization ELISA assaydescribed above. Results indicated that following expansion, all of themaster well supernatants had retained their ability to recognize362.78-CHO antibody (Ab1) in solution. Seven of the master wellsupernatants retained their ability to neutralize the binding of Ab1 tohuman IL-21 ligand.

Cloning: Cells from 5 master wells were chosen according to theirneutralizing activity and cloned in hybridoma growth medium supplementedwith 1×HT (100×, Gibco Invitrogen) in order to isolate a clonalhybridoma producing the neutralizing mAb of interest. Cells were clonedin 96-well microtiter cell culture plates using a standard low-densitydilution (less than 1 cell per well) approach and monoclonality wasassessed by microscopic examination of wells for a single foci of growthprior to assay. Six days post-plating, all plates were screened by theneutralization ELISA for anti-362.78-CHO anti-idiotype inhibitingantibodies. Hybridoma cells from positive wells were successfullyexpanded into 24-well plates.

Selection of First Round Clones: Supernatants from approximately 6 wellsof each cloned hybridoma line that were positive for specific mAb andoriginated from wells with only a single colony of hybridoma growth werecollected from each cloning set and rescreened at various dilutions inthe neutralization ELISA to identify a best neutralizing mAb producingclone. When the density of a best clone of the 24-well cultures wereapproximately 4-6×10⁵ cells/mL, the supernatant was individuallycollected and stored for each well and the cells from each wellcryopreserved.

Summary: mouse monoclonal antibodies (Ab2) reactive against therecombinantly expressed 362.78-CHO antibody (Ab1) were generated andexhibit neutralizing activity capable of blocking the binding of362.78-CHO antibody to human IL-21. These antibodies can be used asreagents in pre-clinical and clinical immunoassays.

Example 19 Binding of Native Intracellular Human and Cynomolgus MonkeyIL-21 (but not Mouse or Rat IL-21) by IL-21 mAb 362.78.1.44

The neutralizing anti-IL-21 monoclonal antibodies described herein weregenerated from transgenic mice expressing human immunoglobulin genes andimmunized with recombinant human IL-21 (see Example 1). It was importantto confirm that the IL-21 mAb clone 362.78.1.44 can bind and neutralizenative human IL-21 in addition to the recombinant form of the IL-21.Additionally, in order to support preclinical toxicology studies, it ishelpful to understand the binding capacity of the IL-21 mAb clone362.78.1.44 to native IL-21 in a variety of species. In order to testthis, one approach is to label IL-21 mAb clone 362.78.1.44 with afluorescent dye and use it to detect intracellular IL-21 in activated Tcells by flow cytometry. In this study, freshly isolated human andcynomolgus monkey peripheral blood leukocytes as well as rat and mousesplenocytes were activated in vitro with PMA and ionomycin for 24 hoursto induce IL-21 production. Cells were harvested, fixed, permeabilizedand stained for expression of CD3 or CD4 (to define helper T cellpopulations) and IL-21 using the IL-21 mAb clone 362.78.1.44 labeledwith ALEXA FLUOR-647 (AF-647), and compared to the staining intensityinduced by an isotype-matched control antibody. A positive signal inthis assay, above that observed for the isotype control mAb,demonstrates specific IL-21 mAb clone 362.78.1.44 binding to endogenousIL-21 in the test species, though it is not an indicator of IL-21neutralizing activity. Further studies are required to demonstrate IL-21neutralization in a species that tests positive for IL-21 binding withthe anti-human IL-21 mAb clone 362.78.1.44 (see Example 20).

Isolation of human PBMC: 100 mL peripheral blood was collected fromhealthy human volunteers (ZymoGenetics) in green top heparin Vacutainertubes (Becton Dickinson, San Jose, Calif.). Blood was diluted with 100mL of room temperature PBS and 35 mL aliquots were distributed into 50mL conical tubes. 14 mL of room temperature Ficoll/Paque PLUS(Pharmacia, Uppsala, Sweden) was underlaid and the tubes were spun for20 minutes at 2000 rpm. The PBMC interface layer was removed and washedtwo times with assay media (RPMI 1640 with supplementalPenicillin/Streptomycin, 10% Fetal Bovine Serum, Sodium Pyruvate, 2 μMβ-Mercaptoethanol, all from Invitrogen, Carlsbad, Calif.). Viable cellswere counted in trypan blue using standard techniques.

Isolation of cynomolgus monkey PBMC: 40 mL peripheral blood wascollected in green-top heparin Vacutainer blood collection tubes (BDBiosciences) from a cymomolgus monkey housed at the University ofWashington in Seattle. Blood was diluted with 40 mL of room temperaturePBS and 35 mL aliquots were distributed into 50 mL conical tubes.Fourteen mL of room temperature Ficoll/Paque PLUS (Pharmacia, Uppsala,Sweden) was underlaid and the tubes were spun for 25 minutes at 2000rpm. The PBMC interface layer was removed and washed two times withassay media (RPMI 1640 with supplemental Penicillin/Streptomycin, 10%Fetal Bovine Serum, Sodium Pyruvate, 2 μM β-Mercaptoethanol). Viablecells were counted in trypan blue using standard techniques.

Isolation of murine and rat splenocytes: Both rat and mouse splenocyteswere prepared according to the following protocol. A freshly collectedspleen was gently disrupted to a single cell suspension using the endsof two frosted glass slides. Cells were then passed through a 70-μMnylon mesh filter to remove clumps. Red blood cells were lysed byresuspending the cell pellet in 2 mL ACK lysis buffer for 10 minutes atroom temperature. This reaction was stopped by the addition of assaymedia, the cells were then centrifuged (1200 RPM for 5 minutes),resuspended and passed over another nylon mesh filter to remove debris.Viable cells were counted in trypan blue using standard techniques.

Overnight activation of cells with PMA and ionomycin: Cells from allspecies were resuspended at 2.0×10e6 cells per mL. One mL of cells wasthen plated with or without the addition of 20 ng/mL PMA and 200 ng/mLionomycin into a 24-well plate and incubated at 37° C. for 20 hours in ahumidified 5% CO2 tissue culture incubator. After 20 hours, 1.0 μL ofGolgiPlug (BD Pharmingen) was added to each well and the cells wereincubated an additional four hours.

Cell harvest and surface stain: Following the 24 tour incubationdescribed above, cells were harvested, washed with cold FACS buffer andplated at 2.0×105-5.0×10e5 cells per well in a 96-well tissue cultureplate (Becton Dickinson and Co., Franklin Lakes, N.J.). Cells were thenstained with 1 μg/mL of one or more of the following antibodies, asappropriate: anti-murine CD4-PE, anti-rat CD3-PE, anti-rat B220-PE, oranti-monkey CD4-PE, or anti-human CD4-PE for 20 minutes on ice. Cellswere then washed two times in PBS in preparation for fixation.

Cell fixation and permeabilization: To fix cells, each cell pellet wasresuspended in 200 μL of 2% paraformaldehyde and incubated at roomtemperature for 5 minutes. Cells were then centrifuged (5 minutes at1200 rpm) and the supernatants aspirated, and the cells were resuspendedin a permeabilization buffer [PBS supplemented with 0.1% saponin(Calbiochem) and 0.5% BSA (Sigma)] for 10 minutes at room temperature.

Intracellular stain: Following fixation and permeabilization, cells werestained with ˜1 μg/mL of one of the following labeled antibodies:anti-mouse IL-21-AF647, anti-human IL-21 clone 362.78.1.44-AF647 (bothproduced at ZymoGenetics) or a comparator anti-human IL-21-AF647antibody from BD Pharmingen Cells were then incubated at roomtemperature in the dark for 40 minutes. After 40 minutes, the cells werewashed twice with FACS buffer (HBSS supplemented with 1% BSA, 2% HumanAB serum and 0.05% HEPES)

Data acquisition and analysis: Upon completion of staining and washing,cells were resuspended in 400 μL FACS buffer and data were collectedusing a FACS Calibur (BD Pharmingen) running CellQuest software. Datawere analyzed using FCS Express data analysis software (De NovoSoftware, Los Angeles, Calif.).

Results: Detection of human IL-21 in PMA+ionomycin stimulated human Tcells: While only 0.015% of the CD3+ T cells stained positive using theisotype control, approximately 9% of the CD4+ T cells stained positivefor IL-21 using the IL-21 mAb clone 362.78.1.44 labeled with AF-647. Thesame fraction of IL-21+ cells was detected using the commerciallyavailable IL-21 mAb from eBiosciences. This demonstrates that the IL-21mAb can bind endogenously produced IL-21 in human CD4+ T cells.

Detection of cynomolgus monkey IL-21 in PMA+ionomycin stimulatedperipheral blood mononuclear cells: Approximately 3.6% of the CD3+ cynoT cells stained positive for IL-21 using the IL-21 mAb clone 362.78.1.44labeled with AF-647, compared to 0.1% positive using the isotypecontrol. This number is higher than that detected using a commerciallyavailable anti-human IL-21 mAb from eBiosciences. This discrepancy maybe due to a weaker binding affinity of the eBiosciences antibody forcynomolgus IL-21. These results demonstrate that the anti-human IL-21mAb clone 362.78.1.44 can bind endogenously produced IL-21 in cynomolgusmonkey CD3+ T cells.

Detection of murine IL-21 in PMA+ionomycin stimulated splenocytes: Usinga rat anti-murine IL-21 monoclonal antibody generated at ZymoGenetics asa positive control, approximately 13.5% of the activated mouse CD4+ Tcells were positive for IL-21. However, as predicted from western blotsand neutralizing bioactivity assays showing that the anti-human IL-21mAb clone 362.78.1.44 does not bind or neutralize mouse IL-21 (seeExamples 3 and 9), the anti-human IL-21 mAb clone 362.78.1.44 labeledwith AF647 did not detect any IL-21-positive cells. This furtherdemonstrates that the anti-human IL-21 mAb clone 362.78.1.44 does notbind to murine IL-21.

Detection of rat IL-21 in PMA+ionomycin stimulated splenocytes: Ratsplenocytes were stimulated overnight in the presence of PMA andionomycin. These stimulation conditions were sufficient to produce IL-21positive T cells in human, cynomolgus monkey and murine T cells. In thisexperiment, neither the anti-mouse IL-21 mAb nor the anti-human IL-21mAb clone 362.78.1.44 detected any cells that were positive for IL-21.However, because there was no positive control in this experiment, thisnegative result does not conclusively eliminate the possibility that theanti-human IL-21 mAb clone 362.78.1.44 can bind to rat IL-21. However,these data, considered along with the lack of neutralization of ratIL-21 bioactivity by the anti-human IL-21 mAb clone 362.78.1.44 in otherassays (see Example 9), does strongly suggest that this mAb probablydoes not bind rat IL-21.

Conclusion: The IL-21 mAb clone 362.78.1.44 described herein clearlybinds to the native human and cynomolgus monkey forms of the IL-21protein but not to murine or rat IL-21.

TABLE 14 Anti-human IL-21 Species Isotype control mAb (clone 78)Anti-IL-21 Positive control Human 0.015% of CD3+ T cells 9% of CD3+ Tcells were 10% of CD3+ T cells were stained positive with an IL-21+IL-21+ (eBiosciences αIL-21 hIgG4-AF647 control mAb*) Cynomolgus 0.1% ofCD3+ T cells 3.6% of CD3+ T cells were 0.2% of CD3+ T cells were Monkeystained positive with an IL-21+ IL-21+* hIgG4-AF647 control Mouse Noisotype control used None detected 13.5% of CD4+ T cells were IL-21+ RatNo isotype control used None detected Not available *(this mAb may notbind strongly to cyno IL-21)

Example 20 Binding and Neutralization of Native Human IL-21 Bioactivityby Clone 362.78.1.44

The neutralizing anti-IL-21 monoclonal antibodies (IL-21 mAb) describedherein were generated from transgenic mice expressing humanimmunoglobulin genes and immunized with recombinant human IL-21 (seeExample 1). It was important to confirm that the IL-21 mAb clone362.78.1.44 can bind and neutralize native human IL-21 in addition tothe recombinant form of IL-21. To demonstrate neutralization of nativeIL-21, the Baf3/IL-21R pSTAT cell-based assay previously described (seeExample 7) was utilized and activated CD4+ T cell conditioned mediasamples were used as the source of native IL-21. In this experiment, Tcell conditioned media samples were preincubated with varying amounts ofIL-21 mAb clone 362.78.1.44 and the level of IL-21-induced STAT3phosphorylation (pSTAT3) in the Baf3/hIL-21R transfectants was thenmeasured. Neutralization of native IL-21 was demonstrated usingactivated T cell conditioned media samples from four separate healthyhuman donors.

Isolation of human PBMC and generation of T cell conditioned mediasamples: 100 mL of peripheral blood was collected from 4 healthy humanvolunteers (ZymoGenetics) in green top heparin Vacutainer tubes (BectonDickinson, San Jose, Calif.). Blood was then diluted with 100 mL of roomtemperature PBS and 35 mL aliquots were distributed into 50 mL conicaltubes. 14 mL of room temperature Ficoll/Paque PLUS (Pharmacia, Uppsala,Sweden) was underlaid and the tubes were spun for 20 minutes at 2000rpm. The PBMC interface layer was removed and washed two times withassay media (RPMI 1640 with supplemental Penicillin/Streptinomycin, 10%Fetal Bovine Serum, Sodium Pyruvate, 2 μM β-Mercaptoethanol, all fromInvitrogen, Carlsbad, Calif.) Viable cells were counted in trypan blueusing standard techniques. T cells were negatively selected using aHuman CD4+ T Cell Selection Kit from Miltenyi Biotec (Auburn, Calif.)following the protocol outlined by the manufacturer. Using standardimmunophenotyping techniques, the CD4+ T cells were subsequentlydetermined to be >95% pure by flow cytometry. T cells were thenincubated for three days at 5×10e5 cells per well in a 24-well platepre-coated with 5.0 μg/mL anti-CD3 antibody in Th1 skewing mediacontaining 5.0 μg/mL anti-IFNγ, 1.0 μg/mL anti-CD28 (all from BectonDickinson) and 10 ng/mL recombinant IL-12 (R&D Systems, Minneapolis,Minn.). After three days, cells were washed, re-plated in mediacontaining 25 ng/mL PMA and 500 ng/mL ionomycin and incubated for fivehours at 37° C. After five hours, conditioned media samples wereharvested and frozen and stored at −80° C. until day of experiment.

To estimate the approximate IL-21 concentration in the T cellconditioned media samples, 1:4 serial dilutions were prepared and testedfor induction of STAT3 phosphorylation in the Baf3/hIL-21Rtransfectants. Following the 10 minute pSTAT3 bioassay protocol outlinedin Example 7, the relative concentration of IL-21 in each conditionedmedia sample was estimated by comparing the level of pSTAT3 to thatgenerated using a titration of recombinant IL-21. Using these data, theconcentration of IL-21 in each of the four conditioned media samples wasdetermined to be between 5.0 and 10.0 ng/mL.

To demonstrate neutralization of native IL-21-induced STAT3phosphorylation, 1:10 dilutions (final IL-21 concentrations between 0.5and 1.0 ng/mL) of the four T cell conditioned media samples werepreincubated for 30 minutes at 37° C. with a 1:4 serial dilution ofIL-21 mAb clone 362.78.1.44. The concentration of clone 362.78.1.44ranged from 0.4 to 400 ng/mL. After 30 minutes, the conditionedmedia+IL-21 mAb samples were transferred to the Baf3/hIL-21R cell plateand incubated for an additional 10 minutes at 37° C. After 10 minutes,the reactions were stopped with cold wash buffer, cells lysed and amountof pSTAT3 measured using the method described in Example 7.

In all four conditioned media samples, the clone 362.78.1.44 IL-21 mAbeffectively neutralized IL-21 activity (data summarized in Table 15).These data clearly demonstrate effective binding to and neutralizationof native human IL-21 by clone 362.78.1.44.

TABLE 15 Neutralization of native IL-21 by IL-21 mAb clone 362.78.1.44IL-21 mAb Conc. pSTAT3 induction (fold over background) (ng/mL) Donor 1Donor 2 Donor 3 Donor 4 0.4 69.49 56.32 61.11 62.73 1.6 70.84 61.7368.24 65.41 6.25 70.76 10.46 7.51 60.65 25 2.16 1.62 1.70 2.49 100 1681.43 1.51 1.49 400 1.59 1.30 1.14 1.51

Example 21

21A. Pilot Toxicity Study with IL-21 mAb 362.78-CHO in CynomolgusMonkeys

The epitope specificity of IL-21 mAb 362.78-CHO is shared by humans,rhesus and cynomolgus macaques (see Examples 17 and 17b), thereforetolerability and toxicity of IL-21 mAb was tested in cynomolgus monkeys,a relevant species for safety assessment.

Cynomolgus monkeys were treated with a single injection of IL-21 mAb362.78-CHO and monitored for clinical signs for 4 to 8 weeks followingtreatment. The IL-21 mAb was delivered by subcutaneous or intravenousinjection at doses of 5 or 100 mg/kg. No clinical signs were observed.No meaningful changes in body weight or coagulation were observed. Nochanges in serum chemistry or hematology attributable to drug toxicitywere observed. The single treatment with IL-21 mAb 362.78-CHO at 5 or100 mg/kg was well tolerated by all of the animals.

Necropsy was performed on the high-dose (100 mg/kg) animals. No grossanatomic changes were observed. Histopathology analysis of the high-doseanimals showed minimal lymphoid hyperplasia in lymphoid tissues.Immunohistochemistry analysis of lymphoid tissues showed a moderateincrease in follicle size and in follicle-associated cell types. Thesechanges could relate to the pharmacological activity of IL-21 mAb362.78-CHO, since IL-21 is known to directly affect the development andegress of B cells from lymphoid follicles and to support class switch,affinity maturation and plasma cell development.

Pharmacokinetic behavior and bioavailability of IL-21 mAb 362.78-CHO wasmonitored in a single dose study in cynomolgus monkeys. Eight malecynomolgus monkeys were treated with IL-21 mAb 362.78-CHO. Three weretreated by subcutaneous injection of 5 mg/kg and three were treated byintravenous injection of 5 mg/kg. Two were treated by intravenousinjection of 100 mg/kg. Serum samples were taken for analysis of IL-21mAb 362.78-CHO levels during four weeks following treatment for the 100mg/kg group and during eight weeks following treatment for the two 5mg/kg groups. Noncompartmental analysis of pharmacokinetic profilesshowed that exposure increased in a dose-proportional manner withintravenous administration of 5 or 100 mg/kg IL-21 mAb. Bioavailabilityof IL-21 mAb 362.78-CHO was approximately 50% following subcutaneousadministration. The estimated terminal half-life for IL-21 mAb was 10-14days. The estimated terminal half-life of IL-21 mAb *insert: 362.78-CHOin cynomolgus monkeys was 10-14 days.

21B. Pilot Pharmacology Study with 362.78-CHO in Cynomolgus Monkeys

The epitope specificity of IL-21 mAb 362.78-CHO is shared by humans,rhesus and cynomolgus macaques (see Examples 17 and 17b), therefore invivo pharmacology of IL-21 mAb was tested in cynomolgus monkeys, arelevant species for pharmacodynamic assessment.

Cynomolgus monkeys were treated with a single injection of IL-21 mAb362.78-CHO and monitored for clinical signs for 4 to 8 weeks followingtreatment. The IL-21 mAb 362.78-CHO was delivered by subcutaneous orintravenous injection at doses of 5 or 100 mg/kg. All animals weremonitored for changes in peripheral blood leukocyte composition by flowcytometry. No treatment-related changes in monocyte or B cellconcentration, and no changes in T cell CD4 and CD8 subsets, nor in theratio of CD4 to CD8 cells, were observed. A reduction in the NK cellconcentration was observed following IL-21 mAb 362.78-CHOadministration. In all treatment groups, peripheral blood NK cells weredecreased at 24 h post-treatment, relative to the baseline values. Infive of the eight animals, NK levels remained below 60% of the baselinevalue for at least two weeks. A confirmatory study with adequatecontrols for handling stress and other sources of variability inperipheral blood NK cell concentration will be required to confirm thisobservation.

Example 22 Anti-mIL-21 Antibody Decreases Disease Incidence andProgression in Mouse Collagen Induced Arthritis (CIA) Model

Mouse Collagen Induced Arthritis (CIA) Model:

There are several animal models for rheumatoid arthritis known in theart. For example, in the collagen-induced arthritis (CIA) model, micedevelop chronic inflammatory arthritis that closely resembles humanrheumatoid arthritis. Since CIA shares similar immunological andpathological features with RA, this makes it an ideal model forscreening potential human anti-inflammatory compounds. The CIA model isa well-known model in mice that depends on both an immune response, andan inflammatory response, in order to occur. The immune responsecomprises the interaction of B-cells and CD4+ T-cells in response tocollagen, which is given as antigen, and leads to the production ofanti-collagen antibodies. The inflammatory phase is the result of tissueresponses from mediators of inflammation, as a consequence of some ofthese antibodies cross-reacting to the mouse's native collagen andactivating the complement cascade. An advantage in using the CIA modelis that the basic mechanisms of pathogenesis are known. The relevantT-cell and B-cell epitopes on type II collagen have been identified, andvarious immunological (e.g., delayed-type hypersensitivity andanti-collagen antibody) and inflammatory (e.g., cytokines, chemokines,and matrix-degrading enzymes) parameters relating to immune-mediatedarthritis have been determined, and can thus be used to assess testcompound efficacy in the CIA model (Wooley, Curr. Opin. Rheum. 3:407-20,1999; Williams et al., Immunol. 89:9784-788, 1992; Myers et al., LifeSci. 61:1861-78, 1997; and Wang et al., Immunol. 92:8955-959, 1995). Thepotential efficacy of a rat anti-mouse IL-21 mAb produced atZymoGenetics was tested in the CIA model, as described below.

Ten-week old male DBA/1J mice (Jackson Labs) were used for therapeuticdosing (i.e. as mice get established disease). On day −21, all animalswere given an intradermal tail injection of 100 microliters of 1 mg/mLchick Type II collagen formulated in Complete Freund's Adjuvant(prepared by Chondrex, Redmond, Wash.), and three weeks later on Day 0they were given the same injection except prepared in IncompleteFreund's Adjuvant. An anti-IL-21 antibody or vehicle (PBS) wasadministered as an intraperitoneal injection every other day for a totalof 6 doses as soon as a mouse developed established disease. Mice (n=7per treatment) received either 0.15 mg of an anti-IL-21 antibody peranimal per dose, or the vehicle control, PBS (Life Technologies,Rockville, Md.). Animals began to show symptoms of arthritis followingthe second collagen injection, with most animals developing inflammationwithin 1-2 weeks. The extent of disease was evaluated in each paw byusing a caliper to measure paw thickness, and by assigning a clinicalscore (0-3) to each paw (see below).

Monitoring Disease:

Animals can begin to show signs of paw inflammation soon after thesecond collagen injection, and some animals may even begin to have signsof toe inflammation prior to the second collagen injection. Most animalsdevelop arthritis within 1-2 weeks of the boost injection, but some mayrequire a longer period of time. Incidence of disease in this model istypically 90-100%, and 0-5 non-responders (determined after 6 weeks ofobservation) are typically seen in a study using 60 animals. Since thisstudy only included mice with established disease, mice that did notdevelop arthritis were not used. Note that as inflammation begins, acommon transient occurrence of variable low-grade paw or toeinflammation can occur. For this reason, an animal was not considered tohave established disease until marked, persistent paw swelling haddeveloped.

All animals were observed daily to assess the status of the disease intheir paws, which was done by assigning a qualitative clinical score toeach of the paws. Every day, each animal had its 4 paws scored accordingto its state of clinical disease. To determine the clinical score, thepaw can be thought of as having 3 zones, the toes, the paw itself (manusor pes), and the wrist or ankle joint. The extent and severity of theinflammation relative to these zones was taken into account including:observation of each toe for swelling; torn nails or redness of toes;notation of any evidence of edema or redness in any of the paws;notation of any loss of fine anatomic demarcation of tendons or bones;evaluation of the wrist or ankle for any edema or redness; and notationif the inflammation extends proximally up the leg. A paw score of 1, 2,or 3 was based first on the overall impression of severity, and secondon how many zones were involved. The scale used for clinical scoring isshown below.

Clinical Score:

0=Normal

0.5=One or more toes involved, but only the toes are inflamed

1=mild inflammation involving the paw (1 zone), and may include a toe ortoes

2=moderate inflammation in the paw and may include some of the toesand/or the wrist/ankle (2 zones)

3=severe inflammation in the paw, wrist/ankle, and some or all of thetoes (3 zones)

Established disease was defined as a qualitative score of pawinflammation ranking 1 or more, that persisted for two days in a row.Once established disease was present, the date was recorded anddesignated as that animal's first day with “established disease”.

Mice receiving an anti-mIL-21 antibody were characterized by areductions in paw swelling over the course of the experiment and had anapproximately 25% lower average arthritis score compared to micereceiving PBS. These results indicate that an anti-mIL-21 antibodyreduced paw swelling and disease progression associated with this modelof arthritis and suggest that an anti-IL-21 antibody may be efficaciousin the treatment of human arthritis.

Example 23 Expression of IL-21R in Human Psoriatic Skin Samples

Expression of IL-21R is generally limited to cells of hematopoieticorigin. However, in settings of inflammatory disease, IL-21R expressionon non-hematopoietic cells may provide a direct stimulus to cell typesthat mediate the functional changes in the affected tissues. Inpsoriasis, keratinocyte growth is dysregulated, with psoriaformepidermal hyperplasia, aberrant terminal differentiation, and incompletedevelopment of the stratum corneum. The IL-21 produced by infiltratingTh1 and Th17 cells in psoriatic skin could promote functional changes inkeratinocytes, including cell proliferation, production of chemokines,and altered differentiation. The presence of IL-21R on keratinocytes inpsoriatic skin lesions was therefore investigated.

Methods:

Immunohistochemistry analysis of 18 skin biopsy samples from 4 normalhuman donors and 9 patients with psoriasis was performed, using a mouseIgG1 antibody against human IL-21R produced at ZymoGenetics. For thepsoriasis patients, a subset (5) provided samples from both lesional andnon-lesional skin. A high degree of epidermal hyperplasia was noted inhistopathology examination of the lesional skin from all donors.Immunoreactivity (staining) of IL-21R on specific cell types was scoredbased on frequency of positive cells and intensity of staining.

Results:

In normal skin and in non-lesional skin of psoriasis patients, stainingfor IL-21R was positive on occasional intra-epidermal mononuclear (MNC),scattered macrophages and fibroblast-like cells. Positive staining forIL-21R was present on high numbers of MNC in all of the lesional samplesfrom psoriasis patients. Samples stained with a mouse isotype controlantibody were negative. Minimal or no staining for IL-21R was present onepidermal keratinocytes from normal skin. In non-lesional skin biopsiesfrom psoriasis patients, mild staining was observed in epidermalkeratinocytes in 4 samples, and moderate staining was observed in thestratum spinosum in the fifth sample. There was mild to strong stainingof focal areas of keratinocytes in the lesional samples from 8 of the 9psoriasis patients.

CONCLUSION

In psoriatic skin lesions, the expression of IL-21R is not limited toinfiltrating leukocytes but is also up-regulated on epidermalkeratinocytes. The presence of increased staining for IL-21R onepidermal keratinocytes in non-lesional skin from psoriasis patients,compared with normal controls, suggests that even in non-involved skin,the keratinocytes may respond aberrantly to IL-21 stimulation. In thepresence of inflammation, increased IL-21R on infiltrating MNC and inthe hyperplastic epidermal layer was noted. Treatment of psoriasis withan IL-21 blocking antibody may therefore inhibit inflammation byblocking IL-21 signals to both inflammatory cells and epidermalkeratinocytes.

TABLE 16 IL-21R Immunoreactivity¹ in Normal, Non-Lesional, and LesionalPsoriatic Skin Skin Type (N) Epidermis² MNC² Normal (4) 0 1 PsoriasisNon-Lesional (5) 1 1 Psoriasis Lesional³ (9) 2 3 ¹Scale: 0 = none, 1 =mild, 2 = moderate, and 3 = strong for IL-21R expression ²Median scoreof all samples tested ³Five of the samples had donor-matchednon-lesional skin biopsies

1. A method of treating a subject having an IL-21-mediated disease, themethod comprising administering to said subject an anti-IL-21 antibody,or antigen binding portion thereof, comprising a heavy chain variableregion comprising amino acid residues 20 to 139 of SEQ ID NO: 61 or alight chain variable region comprising amino acid residues 23 to 129 ofSEQ ID NO: 69, or a heavy chain variable region comprising amino acidresidues 20 to 145 of SEQ ID NO: 29 or a light chain variable regioncomprising amino acid residues 21 to 126 of SEQ ID NO: 37, wherein IL-21activity is inhibited.
 2. The method of claim 1, wherein the antibodycomprises a heavy chain variable region comprising amino acid residues20 to 139 of SEQ ID NO: 61 or a light chain variable region comprisingamino acid residues 23 to 129 of SEQ ID NO:
 69. 3. The method of claim1, wherein the antibody comprises a heavy chain variable regioncomprising amino acid residues 20 to 145 of SEQ ID NO: 29 or a lightchain variable region comprising amino acid residues 21 to 126 of SEQ IDNO:
 37. 4. The method of claim 1, wherein the antibody comprises (a) aheavy chain variable region comprising amino acid residues 20 to 139 ofSEQ ID NO: 61 and a light chain variable region comprising amino acidresidues 23 to 129 of SEQ ID NO: 69, or (b) a heavy chain variableregion comprising amino acid residues 20 to 145 of SEQ ID NO: 29 and alight chain variable region comprising amino acid residues 21 to 126 ofSEQ ID NO:
 37. 5. The method of claim 4, wherein the antibody comprisesa heavy chain variable region comprising amino acid residues 20 to 139of SEQ ID NO: 61 and a light chain variable region comprising amino acidresidues 23 to 129 of SEQ ID NO:
 69. 6. The method of claim 4, whereinthe antibody comprises a heavy chain variable region comprising aminoacid residues 20 to 145 of SEQ ID NO: 29 and a light chain variableregion comprising amino acid residues 21 to 126 of SEQ ID NO:
 37. 7. Themethod of claim 1, wherein the Fc portion of the antibody is selectedfrom the group consisting of IgG1, IgG2, and IgG4.
 8. The method ofclaim 1, wherein the Fc portion of the antibody is modified to reduceeffector functions.
 9. A method of treating a subject having anIL-21-mediated disease, the method comprising administering to saidsubject an anti-IL-21 antibody, or antigen binding portion thereof,which binds to the same epitope as an antibody comprising (a) a heavychain variable region comprising amino acid residues 20 to 139 of SEQ IDNO: 61 and a light chain variable region comprising amino acid residues23 to 129 of SEQ ID NO: 69, or (b) a heavy chain variable regioncomprising amino acid residues 20 to 145 of SEQ ID NO: 29 and a lightchain variable region comprising amino acid residues 21 to 126 of SEQ IDNO: 37, wherein IL-21 activity is inhibited.
 10. The method of claim 9,wherein the isolated monoclonal antibody, or antigen-binding fragmentthereof, binds to the same epitope as an antibody comprising a heavychain variable region comprising amino acid residues 20 to 139 of SEQ IDNO: 61 and a light chain variable region comprising amino acid residues23 to 129 of SEQ ID NO:
 69. 11. The method of claim 9, wherein theisolated monoclonal antibody, or antigen-binding fragment thereof, bindsto the same epitope as an antibody comprising a heavy chain variableregion comprising amino acid residues 20 to 145 of SEQ ID NO: 29 and alight chain variable region comprising amino acid residues 21 to 126 ofSEQ ID NO:
 37. 12. A method of treating a subject having anIL-21-mediated disease, the method comprising administering to saidsubject an anti-IL-21 antibody, or antigen binding portion thereof,comprising (a) a heavy chain variable region comprising the amino acidsequences set forth in SEQ ID NOs: 63, 65, and 67, and a light chainvariable region comprising the amino acid sequences set forth in SEQ IDNOs: 71, 73, and 75, or (b) a heavy chain variable region comprising theamino acid sequences set forth in SEQ ID NOs: 31, 33, and 35 and a lightchain variable region comprising the amino acid sequences set forth inSEQ ID NOs: 39, 41, and 43, wherein IL-21 activity is inhibited.
 13. Themethod of claim 12, wherein the antibody comprises a heavy chainvariable region comprising the amino acid sequences set forth in SEQ IDNOs: 63, 65, and 67, and a light chain variable region comprising theamino acid sequences set forth in SEQ ID NOs: 71, 73, and
 75. 14. Themethod of claim 12, wherein the antibody comprises a heavy chainvariable region comprising the amino acid sequences set forth in SEQ IDNOs: 31, 33, and 35 and a light chain variable region comprising theamino acid sequences set forth in SEQ ID NOs: 39, 41, and
 43. 15. Themethod of claim 12, wherein the Fc portion of the antibody is selectedfrom the group consisting of IgG1, IgG2, and IgG4.
 16. The method ofclaim 12, wherein the Fc portion of the antibody is modified to reduceeffector functions.
 17. The method of claim 1, 12, or 9, wherein thedisease is an autoimmune disease or inflammatory disease.
 18. The methodof claim 17, wherein the disease is selected from the group consistingof inflammatory bowel disease (IBD), rheumatoid arthritis, multiplesclerosis, type I diabetes (IDDM), and lupus.